Atomic therapeutic indicator

ABSTRACT

The present invention relates to the generation of an Atomic Therapeutic Indicator (ATI) for a test sample by the quantification of manganese; in voxels of a 3D region of the sample, wherein the 3D region is topographically defined by co-ordinates X′×Y′×Z. The ATI is used to assess the radio-responsiveness i.e. sensitivity or resistance to radiation treatment, of a cancer i.e. a tumour/neoplasm. In a preferred embodiment, the present invention relates to a method of generating the ATI, assessing the radio-responsiveness of a tumour/neoplasm based on the ATI and, based on the assessment, either treating or not treating the tumour with radiation. The present invention also relates to a method of determining if a cancer is likely to reoccur post radiation treatment comprising quantifying the level of manganese in voxels of a 3D region of a test sample from the cancer and determining the frequency of high metallomic regions (HMRs) in the cancer, wherein a high frequency of HMRs is indicative that the cancer is likely to reoccur and a low frequency of HMRs is indicative that the cancer is unlikely to reoccur; and associated methods of treatment.

FIELD OF THE INVENTION

The present invention relates to the generation of an Atomic TherapeuticIndicator (ATI) for a test sample by the quantification of manganese; invoxels of a 3D region of the sample, wherein the 3D region istopographically defined by co-ordinates X′xY′xZ. The ATI is used toassess the radio-responsiveness i.e. sensitivity or resistance toradiation treatment, of a cancer i.e. a tumour/neoplasm. In a preferredembodiment, the present invention relates to a method of generating theATI, assessing the radio-responsiveness of a tumour/neoplasm based onthe ATI and, based on the assessment, either treating or not treatingthe tumour with radiation.

The present invention also relates to a method of determining if acancer is likely to reoccur post radiation treatment comprisingquantifying the level of manganese in voxels of a 3D region of a testsample from the cancer and determining the frequency of high metallomicregions (HMRs) in the cancer, wherein a high frequency of HMRs isindicative that the cancer is likely to reoccur and a low frequency ofHMRs is indicative that the cancer is unlikely to reoccur; andassociated methods of treatment.

The invention further relates to a method of determining theradio-responsiveness of a melanoma, the method comprising determiningthe level of melanin in a test sample from the melanoma, wherein thelower the level of melanin the more sensitive the melanoma is toradiation and the higher the level of melanin the more resistant themelanoma is to radiation; and associated methods of treatment.

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should inno way be considered as an admission that such prior art is widely knownor forms part of common general knowledge in the field.

Over 60% of patients in the USA will receive radiation treatment duringan illness, and these 600,000 or so individuals will make over 20million radiation therapy visits, with each patient receiving, onaverage, 30 treatments of external beam radiation therapy with curativeintent. Breast, lung and prostate cancer patients make up over 50% ofall patients receiving radiation therapy (American Society for RadiationOncology, ASTRO, 2012).

The clinical decision to treat “cancer patients” with radiation, or toavoid the use of this therapeutic modality, is currently based on amixture of subjective, empirical and historical practice, commonlyencapsulated within medical “art”. The treatment and managementdecisions initially involve the suitability of the patients fordefinitive surgery, their age, extent of cardiopulmonary reserve,defined co-morbidities, the pathological grade of the tumour, and thestage of the disease as determined by various imaging modalities such asMRI, fMRI, CT, ultrasound, X-ray, PET, and combinations thereof. Thereis minimal quantitative input in terms of the properties of the tumouritself, with pathologists interpreting how tumourous cells “look” interms of their morphological deviation from normal cells, thedifferences that are apparent in tumor architecture from that of normaltissues, and the extent of cell division within microscopic fields.

More specifically, the assessment of whether an individual is suspectedof having a tumour or a cancer that requires treatment currently dependsupon pathological examination of a sample of tissue, organ or bloodsample. The results of the pathology uniquely determine the flow-onclinical events that are part of the current conveyor belt of medicalart. In the United States of America (USA), more than 60% of cancerpatients receive some form of radiation treatment, usually incombination with surgery and systemic therapies involving selecteddrugs, and more recently in combination with immunotherapies. Thecritical decision on whether a patient should, or should not, receiveradiation, depends first and foremost on the report made by apathologist (Davidson and Rimm; JAMA; 313; 1109-1110; 2015. Elmore etal., JAMA; 313; 1122-1132. 2015). This report is the current basis uponwhich a determination of patient management is made, which is usuallycarried out in a multidisciplinary setting involving radiationoncologists, medical oncologists and surgeons. However, such acritically important decision currently relies on a pathologicaldiagnosis primarily based on the subjective analysis also describedabove, concerning cellular morphology and tissue architecture offormalin-fixed paraffin-embedded and Hematoxylin and Eosin (H&E) stainedtissue sections (see FIG. 1, for example).

The concordance among pathologists of diagnostic interpretation isvariable, and depends on the type of tumour under investigation. In thecase of atypical hyperplasia of the breast, the concordance is only 48%(Elmore et al., JAMA, 313; 1122-1132. 2015). In terms of tumours of thethyroid, for example, it is very difficult to discriminate betweennormal tissue and “cancerous” tissue, except with very late stagetumours. This subjectivity of interpretation of the pathology results inovertreatment of those patients who achieve little benefit fromradiotherapy, but suffer its well known harms, and results in undertreatment of those patients who could benefit from radiation, but do notreceive it. To avoid legal issues, physicians err on the side ofcaution, and radiation is given to many patients in the absence of anyquantitative evidence of benefit, in the belief that following surgery,for example, radiation will minimize the effects of any residual cancercells that have been left behind in the margins surrounding the resectedtumour. For those patients whose tumours and associated stromal nicheshave some degree of radio-resistance, the radiation therapy is futile,since even if some radiation sensitive cells are killed, the survivingtumour and stromal cell populations have been selected for even moreradio-resistance.

Medical practitioners generally rely upon evidence-based clinicaldecision support resources such as http://www.uptodate.com. Thisresource provides a current clinical summary of radiation therapytechniques in cancer control at multiple levels, including types ofradiation such as external beam radiotherapy, brachytherapy,intraoperative radiotherapy and targeted radionuclide therapy.

The Coverage and Analysis Group at the Centers for Medicare and MedicaidServices (the federal agency within the US Department of Health andHuman Services) requested an assessment report on prostate cancer thatwas provided by Ip et al., 2010, at the Tufts Evidence-based PracticeCenter under contract to the Agency for Healthcare Research and Quality(RHQ), Rockville, Md., USA. (contract #290 2007 10055 I). This reportaddressed the evidence for the clinical and biochemical outcomes ofdifferent radiotherapies, such as stereotactic body radiation therapy,fractionated external beam radiation therapy and brachytherapy, onpatients with localized prostate cancer (T1 and T2 disease). Theexternal beam radiation therapies (EBRT) include intensity-modulatedradiotherapy, conformal radiation, stereotactic body radiation,CyberKnife and proton beam radiation, while brachytherapy includespermanent implantation of radioactive isotopic “seeds” as well astemporary high dose radioactivity seeds.

The type of radiation delivered to a patient was also evaluated, whetherit was photon or proton based, and whether the radiation was deliveredvia a linear accelerator, gamma rays (from a Cobalt-60 source), or viaradioactive seeds comprising ¹²⁵Iodine, ¹³¹Cesium or ¹⁰³Palladium, forLow Dose Rate Brachytherapy (LDRBT); or ¹⁹²Iridium (for High Dose RateBrachytherapy (HDRBT). The evaluation also included the parameters thatimpinged on patient outcomes including radioactive dosages, adverseevents, treatment planning algorithms, and the number of fractionsdelivered.

The rating system used by Ip et al. to evaluate the various clinicaltrials in terms of the strength of evidence emerging from any trial, wasa subjective 3-tier one: high, moderate and insufficient.

In terms of the benefits versus harms of different radiotherapies, theresults were as noted below.

In terms of the comparison of the benefits versus harms ofradiotherapies, versus no radiation treatment, the strength of theevidence was found to be of category 3, insufficient.

The strength of the evidence was found to be insufficient for patientsurvival, when low dose rate brachytherapy was compared to external beamradiation therapy.

The strength of the evidence was found to be insufficient forbiochemical control, when brachytherapy was compared to external beamradiation therapy and when high dose rate brachytherapy was compared tolow dose rate brachytherapy.

The strength of the evidence was found to be insufficient forgenitourinary and gastrointestinal toxicities, when low dose ratebrachytherapy was compared to external beam radiation therapy.

The strength of the evidence was found to be insufficient for variouscombination therapies, LDRBT plus EBRT.

The strength of the evidence was found to be insufficient for differentstudies within the Stereotactic Body RadioTherapy (SBRT) and EBRTumbrellas, namely bladder and rectal toxicities, freedom frombiochemical failure and genitourinary or gastrointestinal toxicities.

The strength of the evidence was found to be insufficient for low doserate brachytherapy in terms of radioactive seed comparisons, ¹²⁵Iodineand ¹⁰³Palladium.

The strength of the evidence was found to be insufficient for thecontribution of age, race, ethnicity, co-morbidities, treatment-relatedadverse effects and disease progression to the baseline risk of apatient as a contributor to the outcome from radiotherapy.

The detailed report of Ip et al., 2010, concluded that in one of the twomost extensively studied tumour types (localized prostate and localizedbreast), the evidence for the benefits of radiotherapy compared to notreatment for men with T1 or T2 prostate “cancer” revealed noquantitative indicators for radiation treatment of patients.Furthermore, there was substantial heterogeneity within and betweenstudies, with many of the findings in this large evaluation beinginconsistent.

The Ip et al. report indicates that the underlying risk of progressionof the disease to the metastatic state varies widely between patients.The inability to objectively determine risk of progression means thatpatients deemed to be at “low” risk are advised to undergobrachytherapy, whereas those deemed to be at “intermediate” risk tend tobe given external beam radiotherapy.

There is therefore an urgent need to identify those patients who have abiological parameter that favours one treatment modality compared withanother, e.g., tumour characteristics that are favourable for radiationtreatment, namely those whose tumours are sensitive to radiation versusthose patients whose tumours are more radiation resistant, and thereforeshould be spared radiation treatment which is likely to be futile andharmful.

There is a further need for identifying biological parameters that mightbe useful in distinguishing characteristics of the abnormal tumour cellsthemselves, and the characteristics of the stroma, and the threedimensional (3D) distribution in which such abnormal cells are embedded.For example, a tumour that has abnormal cells evenly spread within astromal component, is very different to a tumour where the abnormalcells are largely separate from stromal cells. In the case of prostatecancer, both of these situations occur within different foci of abnormalcells within the gland itself and also within metastases to bone. Nocurrent external imaging methods (MRI, CT or ¹⁸FDG imaging methods) canreliably identify these different areas, or their differentcharacteristics.

To date, there are no available data pertaining to the use ofradiotherapy for a given tumour of a particular patient: current datahave no solid quantitative basis.

There remains a need for assays that provide quantitative indicatorsthat enable the identification of a biological parameter in a sample,e.g., radio-sensitivity or radio-insensitivity/radio-resistance whenmaking a suitable decision in respect of treating patients such aswhether or not to treat “cancer patients” with radiation, or to avoidthe use of radiation.

It is an objective of the present invention to overcome or ameliorate atleast one of the disadvantages of the prior art treatments or/and toprovide a useful alternative.

SUMMARY OF THE INVENTION

The inventor of the present invention has surprisingly found that thelevel of manganese in a cancer can be used as an indicator ofradio-responsiveness of the cancer. In particular, the higher the levelof manganese in a cancer the more resistant the cancer to radiation; andthe lower the level of manganese in the cancer, the more sensitive thecancer to radiation. Radio-responsiveness of a cancer can best bedetermined using a combination of 3D and 2D analysis. Accordingly, inone aspect, the present invention provides a method of generating anAtomic Therapeutic Indicator (ATI) of a biological test samplecomprising quantifying the level of manganese in voxels in a 3D regionof the test sample, wherein the method comprises:

-   -   (a) selecting a 2D region of said test sample, wherein the 2D        region is topographically defined by an X′:Y′ coordinate system        wherein X′ is the length of the 2D region and Y′ is the breadth        of the 2D region, wherein the 3D region corresponds to said 2D        region and has a selected height represented by Z, wherein the        3D region is divided into voxels of a pre-defined volume, the        volume of each voxel being defined by X×Y×Z wherein X is the        length of the voxel, Y is the breadth of the voxel and Z is the        height of the voxel;    -   (b) quantifying the level of manganese in each voxel; and    -   (c) calculating the central tendency level of manganese in        selected voxels;        wherein the central tendency level of manganese in the selected        voxels defines the ATI.

In another aspect the present invention provides a method of determiningthe radio-responsiveness of a cancer, the method comprising generatingan ATI of a test sample according to the invention, wherein the lowerthe ATI the more sensitive the cancer is to radiation and the higher theATI the more resistant the cancer is to radiation.

In one embodiment, the ATI is compared to a pre-determined ATI thresholdwherein the radio-responsiveness of the cancer is determined byassessing whether the ATI is above or below the ATI threshold, and

-   -   wherein if the ATI is below the ATI threshold the cancer is        determined to be sensitive to radiation; and    -   wherein if the ATI is above the ATI threshold the cancer is        determined to be resistant to radiation.

In another embodiment, the ATI is compared to two pre-determined ATIthresholds wherein the radio-responsiveness of the cancer is determinedby assessing whether the ATI is above or below the two thresholds, and

-   -   wherein if the ATI is below the lower ATI threshold the cancer        is determined to be sensitive to radiation;    -   wherein if the ATI is above the second ATI threshold the cancer        is determined to be resistant to radiation; and    -   wherein if the ATI is between the two ATI thresholds the cancer        is determined to be partially sensitive to radiation.

Preferably, quantification of the level of manganese in the voxels ofthe test sample is calibrated using a reference standard, wherein thereference standard comprises one or more reference voxels, and whereineach reference voxel comprises a known quantity of manganese.

In addition to the known quantity of manganese, the reference standardmay comprise any additional material that allows the quantity ofmanganese in the reference standard to be compared with the quantity orlevel of manganese in the test sample. Preferably the reference standardis a biological sample comprising a known quantity of endogenous orexogenous manganese. The biological sample may comprise human or animaltissue.

Preferably, the reference standard comprises tissue from an animal(including, for example, mammalian or avian species) to which a a knownand amount of manganese is added. The skilled addressee will understandthat matrix-matched reference standards with precisely defined amountsof manganese are useful for calculation of a calibrated counts persecond correction factor to mitigate day-to-day signal variability. Inone embodiment, the reference standard tissue is derived from chickenbreast

In one embodiment, the level of manganese quantified in a 3D region ofone or more control sample(s) is quantified concurrently, orsequentially in any order, when the test sample is being quantified, orside-by-side with the test sample.

In one embodiment, the control sample is added to the test sample foranalysis.

Preferably, the central tendency level is the median, arithmetic mean ormode.

It will be understood by the skilled addressee that the test sample maybe stained or unstained. Preferably, the selected voxels are voxels inwhich cancer cells are detected. More preferably, the cancer cells aredetected by visual inspection of the 2D region of the test samplestained with a stain that distinguishes cancer cells from other cells.When the cancer cells are detected by visual inspection of the 2D regionof the test sample stained with a stain that distinguishes the cancercells from other cells, preferably the stain is hematoxylin and eosin(H&E). In one embodiment, the cancer cells are detected by binding of anantibody, preferably a metal-labelled antibody, to the cancer cells.

In the context of the present invention, the length of the voxel, X, isin any range measurable and preferably in the range of about 1 micron toabout 200 microns. In some embodiments, X is selected from about 10 toabout 50 microns and any value in between, and preferably X is about 35microns.

In the context of the present invention, the breadth of the voxel, Y, isin any range measurable and preferably in the range of about 1 micron toabout 200 microns. In some embodiments, Y is selected from about 10 toabout 50 microns and any value in between, and preferably Y is about 35microns.

In the context of the present invention, Z is in any range measurableand preferably in the range of about 1 micron to about 20 microns.Preferably Z is selected from about 1 to about 20 microns and any valuein between, preferably Z is about 1, or about 2, or about 3, or about 4,or about 5 microns.

In a preferred embodiment, X, Y and Z are selected respectively inranges from about 1 to 200 microns, 1 to 200 microns, and 1 to 20microns and any value in between in each range. Preferably X, Y and Zare 35 microns, 35 microns and 5 microns respectively.

In one embodiment of the invention, the pre-defined volume of each voxelis in the range of about 1 cubic micron to about 8×10⁵ cubic microns, orabout 1 cubic micron to about 10,000 cubic microns, or about 2000 cubicmicrons to about 8,000 cubic microns. In a preferred embodiment, thepre-defined volume is about 6,125 cubic microns.

The level of manganese in the voxels can be determined using anyelemental analysis technique. Preferably, the elemental analysistechnique is laser ablation-Inductively coupled plasma-mass spectrometry(LA-ICP-MS), laser ablation-time-of-flight-mass spectrometry(LA-TOF-MS), inductively coupled plasma-optical emission spectroscopy(ICP-OES), microwave plasma-atomic emission spectroscopy (MP-AES), laserinduced break down spectroscopy (LIBS), secondary ion mass spectrometry(SIMS), or X-ray absorption near edge structure (XANES), atomicabsorption spectroscopy (AA), or X-ray fluorescence (XRF).

The data obtained may be analyzed using computer assisted screeningtechnology. For example, a five micron section may be laser ablated byrastering the laser across the slide laterally, one ablation track at atime from top to bottom. Alternatively, a five micron section may belaser ablated by rastering the laser across the slide from top tobottom, one ablation track at a time laterally. For example, an ATIaccording to the invention may be generated by laser ablation of atleast one track across the test sample and/or control(s), wherein theablation track comprises at least one voxel. In another example, themethod of the invention comprises more than one ablation track andseveral voxels.

Preferably, the test sample is selected from a cell, a population ofcells, one or more single celled organism(s), a tissue sample, or partthereof, an organ sample or part thereof, one or more cellsobtained/derived from a prokaryotic or eukaryotic organism, a populationof cells and its associated non-cellular stromal components, aneoplastic cell or population of neoplastic cells, a tissue sample fromany organ or tissue from a subject, a tumour, a solid mass or a “liquid”population of cells, cells of any cancer of the hematopoietic systemincluding leukemic cells, the circulating cellular derivatives of solidtumours, and a cell or population of cells that has metastasized.

In another aspect, the present invention provides a method of treatingcancer in a subject comprising performing the method of the invention ona test sample from the subject, and including radiotherapy in treatingsaid cancer in the subject if the cancer is determined to be sensitiveto radiation.

In another aspect, the present invention provides a method of treatingcancer in a subject comprising performing the method of the invention ona test sample from the subject, and not including radiotherapy intreating said cancer in the subject if cancer is determined to beresistant to radiation.

Preferably, the subject comprises a normal human or mammalian subject, anormal human or mammalian subject in need of a treatment or prophylaxis,a subject diagnosed with a cancer, a neoplasm/tumour suspected of havinga cancer/neoplasm, a subject undergoing treatment and/or prophylaxis forany disorder including any cancer, an asymptomatic subject that hasundergone a test or scan indicative of an underlying condition, asymptomatic subject that has undergone a test or scan indicative of anunderlying condition, a subject undergoing clinical treatment includingcancer therapy, or clinical intervention in the form of drugs,chemotherapy, immunotherapy, surgery, radiation or therapeutic devices,or a subject not yet undergoing any clinical treatment.

Preferably, the control sample comprises or is derived from a cell, apopulation of cells, one or more single celled organism(s), a tissuesample, or part thereof, an organ sample or part thereof, one or morecells obtained/derived from a prokaryotic or eukaryotic organism, apopulation of cells and its associated non-cellular stromal components,a neoplastic cell or population of neoplastic cells, a tissue samplefrom any organ or tissue from a subject, a tumour wherein the tumour forexample, is a solid mass or a “liquid” population of cells, any cancerof the hematopoietic system including leukemic cells, the circulatingcellular derivatives of solid tumours, or a cell or population of cellsthat has metastasized.

In one embodiment, the test and/or the control sample comprises or isderived from a cell, a population of cells or a tissue sample of atumour/neoplasm of breast cancer, prostate cancer, cancer of the testesincluding seminoma, lymphoma including B-cell lymphoma, small cell lungcancer, cancer of the brain including glioblastoma multiforme,mesothelioma, or melanoma.

In another aspect, the present invention provides a method ofdetermining the likelihood of reoccurrence of a cancer post radiationtreatment comprising:

-   -   (a) quantifying the level of manganese in a 3D region of a test        sample from the cancer by selecting a 2D region of said test        sample, wherein the 2D region is topographically defined by an        X′:Y′ coordinate system wherein X′ is the length of the 2D        region and Y′ is the breadth of the 2D region, wherein the 3D        region corresponds to said 2D region and has a selected height        represented by Z, wherein the 3D region is divided into three or        more voxels of a pre-defined volume, the volume of each voxel        being defined by X×Y×Z, wherein X is the length of the voxel, Y        is the breadth of the voxel and Z is the height of the voxel,        and quantifying the level of manganese in each voxel;    -   (b) identifying in the 2D region corresponding to the X and Y        coordinates of the voxel, high metallomic regions (HMRs), being        regions in which the level of manganese is higher than in the        surrounding areas as enabled by statistical thresholds that are        multiples of a central tendency or any approximation between        integers;        wherein the higher the frequency of HMRs the higher the        likelihood of the cancer reoccurring and the lower the frequency        of HMRs the lower the likelihood of the cancer reoccurring.

Preferably, the HMRs are also identified in the 2D region of the testsample stained with a stain that distinguishes cancer cells from stromalcomponents, and preferably the stain is hematoxylin and eosin (H&E)stain.

In the context of the present invention, radio-responsiveness is ameasure of sensitivity/resistance to radiation treatment.

In one example, the test sample is determined to be sensitive toradiation treatment if the ATI of the test sample is below apre-determined threshold value obtained with the control sample that isknown to be radiation sensitive. In another example, the test sample isdetermined to be resistant to radiation treatment if the ATI of the testsample is above a pre-determined threshold value obtained with thecontrol sample that is known to be radiation resistant.

In one embodiment of the invention, test samples with an ATI at or belowa lower threshold limit are determined to be sensitive to radiationtreatment, and test samples at or above a higher threshold limit aredetermined to be resistant to radiation.

The volume of the voxels comprising the reference standard or thecontrol sample can be the same or different to the volume of the voxelsof the test sample.

The present invention further provides a method according to theinvention herein, wherein an ATI is generated by quantifying the levelof manganese in more than one 3D region of the test sample, andcalculating the central tendency level of manganese across all of the 3Dregions to thereby generate a further ATI.

The present invention further provides a method according to theinvention herein, wherein an ATI is generated by quantifying the levelof manganese in more than one test sample, calculating the centraltendency level of manganese across the test samples to thereby generatea further ATI.

The present invention further provides a method according to theinvention herein, wherein the level of manganese quantified in a 3Dregion of the control sample is quantified concurrently, or sequentiallyin any order, when the test sample is being quantified, or side-by-sidewith the test sample.

It is also contemplated that the control sample may be added to the testsample for analysis.

The Atomic Therapeutic Indicator (ATI) may be expressed in any units.For example, it may be expressed in calibrated counts/second (CC/S), oran equivalent proportional concentration unit, such as micrograms/gram,milligrams/kilogram, parts per million, micrograms/voxel,milligrams/voxel, moles or moles/voxel.

Until the present invention, it was not realized that the ATI from aradiation sensitive sample, e.g., cell/tissue, neoplastic cell/tumour,can be distinguished from the ATI of a sample that is radiationresistant, e.g., cell/tissue, neoplastic cell/tumour. This quantitativedistinction enables the determination of radio-responsiveness (i.e. adetermination of radiation sensitivity and/or radiation resistance) fora given selected cell, tissue sample or part thereof, neoplastic cell,or tumour sample or part thereof. It will be apparent to a personskilled in the art that the term “cell” includes neoplastic cell and theterm “tissue sample or part thereof”, includes a tumour sample or partthereof. The term “neoplastic” as used herein, includes any change in acell that contributes to the potential to give rise to an abnormalgrowth of cells, whether pre-cancerous or cancerous.

The term “tumour” as used herein includes, but is not limited to, acollection of one or more neoplastic cells and/or its associated stromalcomponent in that niche. For example, in the case of metastatic prostatecancer to the bone, one radiation treatment used prior to the presentinvention, is the use of ²²³Radium which has as its preferred target,hydroxyapatite (Ca₅ (PO4)₃ OH), a major bone component. ²²³Radiumefficiently “homes” to bone. When prostate, breast or any other cancercells metastasize to bone, the cancer cells become intermixed withhydroxyapatite. The emission of the short range alpha particle destroysosteoblasts, but does not directly affect the cancer cells per se. Hencein this case, suppression of factors produced by the stroma(osteoblasts), which normally allow prostate cancer cells to grow inthis niche, is disrupted, and the cancer cells do not grow as well, andsurvival of the patient is increased. Accordingly, a person skilled inthe art will understand that different stromal niches support cancercells to a different extent, and those niches themselves may also varyin their metallomic content, all the way from very high to very low andmay also be radio-sensitive or radio-resistant. Thus when radiation isapplied to a “tumour”, there are four boundary condition-typepossibilities. If both stromal and cancer cells are radio-resistant,then radiation to the “tumour” is ineffective and the tumour can undergofurther growth. If both stromal and cancer cells are radio-sensitive,then the growth of a tumour is halted. If the stromal cells areradio-sensitive, and the cancer cells are radio-resistant, the stromalcells are effectively killed by radiation, and the growth of the tumourcells is also halted, because they now have no metabolic/factor supportfrom their stroma, and so the tumour does not grow. If stromal cells areradio-resistant, and the cancer cells are radio-sensitive, tumour growthis halted. Accordingly, a person skilled in the art will understand thatthe term “tumour or part thereof” includes tumour cells and/orassociated stromal cells in their complete diversity (blood vessels,infiltrating and resident immune cells, fibroblasts, pericytes andinfiltrating exosomes).

Determining the level of manganese in a selected 3D region according toany aspect, embodiment or example described herein includes, but is notlimited to, performing a direct measurement on the test sample,reference standard and/or control sample, e.g., the cell, tissue sampleor part thereof, neoplastic cell, tumour sample or part thereof. In oneexample, the test sample and/or control sample may be directly processedin its natural environment, e.g., by direct scanning and quantifying thelevels of manganese in a selected 3D region of the test sample and/orcontrol sample according to known methods. In another example, the testsample and/or control sample comprising or derived from e.g., a cell,tissue sample or part thereof, neoplastic cell, tumour sample or partthereof is prepared for microscopic examination or for automatedanalysis by machine scanning according to known methods. The cell,tissue sample or part thereof, neoplastic cell, tumour sample or partthereof may be flash frozen, or formalin-fixed and paraffin-embedded, orone or more cells are deposited as a monolayer or near monolayer on amicroscopic slide, e.g., via a SurePath-like system. The tissue/tumoursample or part thereof, may also be treated whereby a cell or cellpopulation is obtained therefrom and deposited as a monolayer or nearmonolayer on a microscopic slide, e.g., via a SurePath-like system.Sections of the cell, tissue sample or part thereof, neoplastic cell,tumour sample or part thereof are then treated according to knownmethods to prepare the section for microscopic examination or forautomated analysis by machine scanning. Optionally, one or moresection(s) are stained with H&E stain and/or specific antibodies tovisualize morphological aspects of interest. In one example, one or moresequential sections are fixed unstained and directly used for thedetermination of the level of manganese according to the invention. Inanother example, one or more section(s) are sequentially prepared e.g.,matched, and at least one is stained with H&E stain and/or specificantibodies to visualize morphological aspects of interest and thestained sections are prepared alongside unstained sections, e.g.,sequential sections are prepared, wherein at least one may be stained asdescribed herein, and at least one is unstained. The stained section maybe first visualized then directly used for the determination of thelevel of manganese according to the invention, or a stained section isprepared according to standard methods in the art, and visualized by apathologist to determine morphological aspects, and a matched unstainedsection, e.g., sequential section, is chosen and used for thedetermination of the level of manganese according to the invention.

The level of manganese according to the invention is determined by anymethod known in the art that enables the selected 3D region to betopographically defined by an X′:Y′:Z coordinate system and divided intovoxels of pre-determined volume. For example, the level of manganese ismeasured in a voxel of a pre-defined volume and the same or differentpre-defined volume may be used for the reference standard or the controlsample.

A person skilled in the art will understand that the ATI may be obtainedfor several ablation tracks comprising more than one voxel. For example,the number of ablation tracks may be any number that is conceivable tobe processed and includes, but is not limited to, about 1 to 100ablation tracks. In one example, the number of ablation tracks is atleast 3. The length of the ablation track may also vary and comprisesany number of voxels according to the invention.

A person skilled in the art will appreciate that the number of voxelswill vary based on the voxel size. For example, the track length may beany length the instrumentation used permits. In one example, the tracklength may be between 0.5 to 1.0 cm. In another example, the tracklength is about 5.0 cm, or about 5.35 cm in length. For example, asingle track wherein each voxel is about35(length)×35(breadth)×5(height) with a volume of 6,125 cubic microns,and will yield approximately 1,500 voxels of analysis based on a tracklength of 5.35 cm. It is also contemplated that according to theinvention, one or more areas of a sample is ablated to generate an ATIaccording to any aspect, embodiment or example herein. For example, aperson skilled in the art will appreciate that a 1 mm×1 mm area istypically used by pathologists for measuring mitotic rate on a slide andsuch an area may also be ablated according to the invention. In thisexample, such an area corresponds to an approximately 30 voxel by 30voxel area wherein each voxel is about 35(length)×35(breadth)×5(height)with a volume of 6,125 cubic microns. It is further contemplated thatdefining a minimal area for analysis will depend upon the sensitivity ofthe detection method. For example, the size of the area may vary from 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 etc contiguous voxels, to an integerwhich is the sample size chosen for analysis of that particular tumour.A person skilled in the art will appreciate that size area according tothe invention is in any range measurable using existing technology.

It will also be understood that determining the level of manganeseaccording to the invention may be corrected for background. For example,in the case of laser ablation mass spectrometry, a region without thetest sample and/or control sample may be used for background correction.For example, a test sample and/or control sample on a slide may be laserablated directly, and the background in an area of the slide without thetest sample and/or control is used for background correction.

In one or more embodiments that background correction is set to a levelthat allows for regions of particularly high metallomic content to berevealed. These regions are referred to as high metallomic regions(HMRs).

A person skilled in the art will appreciate that the voxel of the testsample, reference standard and/or control sample according to theinvention is in any range measurable using relevant technology. Forexample, X is in any range measurable and, as indicated above, ispreferably in the range of about 1 micron to about 200 microns and anyvalue in between. In one example, X is selected from about 10 to about50 microns and any value in between. Preferably, X is about 35 microns.For example, Y is in any range measurable and preferably in the range ofabout 1 micron to about 200 microns and any value in between. In oneexample, Y is selected from about 10 to about 50 microns and any valuein between. Preferably, X is about 35 microns. For example, Z is in anyrange measurable and preferably in the range of about 1 micron to about200 microns and any value in between. In one example, Z is selected fromabout 1 to about 20 microns and any value in between. Preferably, Z isabout 1, or about 2, or about 3, or about 4, or about 5, or about 6, orabout 7, or about 8, or about 9, or about 10 microns. A person skilledin the art will appreciate that the X′ and Y′ coordinates define theselected 2D area of the test sample and Z represents the thickness ofthe test sample. Accordingly, as indicated above, the voxel according tothe invention e.g., X×Y×Z, includes, but is not limited to, a rangeselected from about 1 to 200:1 to 200:1 to 20 cubic microns and anyvalue in between. For example, the X value is about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, or 200 microns or any approximation between integers. Forexample, the Y value is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200microns or any approximation between integers. For example, the Z valueis about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 microns or any approximation between integers. Accordingly,any one of each X value listed may be combined with any one of each Yvalue listed, which may then be combined within any one of each Z valuelisted. It will also be understood that X, Y, and Z may not be equal inlength. Alternatively, X, Y and Z may be equal in length. In anotherexample, X and Y are equal in length and Z is not equal to X and Y. Inanother example, X and Z are equal in length and Y is not equal to X andZ. Preferably, X and Y are equal in length. For example, the voxel isabout 1×1×1, or about 5×5×1, or about 10×10×1, or about 15×15×1, orabout 20×20×1, or about 25×25×1, or about 30×3033 1 or about 35×35×1 orabout 40×40×1 or about 45×45×1 or about 50×50×1 or about 55×55×1 orabout 60×60×1 or about 65×65×1 or about 70×70×1, or about 1×1×2, orabout 5×5×2, or about 10×10×2, or about 15×15×2, or about 20×20×2, orabout 25×25×2, or about 30×30×2 or about 35×35×2 or about 40×40×2 orabout 45×45×2 or about 50×50×2 or about 55×55×2 or about 60×60×2 orabout 65×65×2 or about 70×70×2, or about 1×1×3, or about 5×5×3, or about10×10×3, or about 15×15×3, or about 20×20×3, or about 25×25×3, or about30×30×3 or about 35×35×3 or about 40×40×3 or about 45×45×3 or about50×50×3 or about 55×55×3 or about 60×60×3 or about 65×65×3 or about70×70×3, or about 1×1×4, or about 5×5×4, or about 10×10×4, or about15×15×4, or about 20×20×4, or about 25×25×4, or about 30×30×4 or about35×35×4 or about 40×40×4 or about 45×45×4 or about 50×50×4 or about55×55×4 or about 60×60×4 or about 65×65×4 or about 70×70×4, or about1×1×5, or about 5×5×5, or about 10×10×5, or about 15×15×5, or about20×20×5, or about 25×25×5, or about 30×30×5 or about 35×35×5 or about40×40×5 or about 45×45×5 or about 50×50×5 or about 55×55×5 or about60×60×5 or about 65×65×5 or about 70×70×5, or about 1×1×6, or about5×5×6, or about 10×10×6, or about 15×15×6, or about 20×20×6, or about25×25×6, or about 30×30×6 or about 35×35×6 or about 40×40×6 or about45×45×6 or about 50×50×6 or about 55×55×6 or about 60×60×6 or about65×65×6 or about 70×70×6, or about 1×1×7, or about 5×5×7, or about10×10×7, or about 15×15×7, or about 20×20×7, or about 25×25×7, or about30×30×7 or about 35×35×7 or about 40×40×7 or about 45×45×7 or about50×50×7 or about 55×55×7 or about 60×60×7 or about 65×65×7 or about70×70×7, or about 1×1×8, or about 5×5×8, or about 10×10×8, or about15×15×8, or about 20×20×8, or about 25×25×8, or about 30×30×8 or about35×35×8 or about 40×40×8 or about 45×45×8 or about 50×50×8 or about55×55×8 or about 60×60×8 or about 65×65×8 or about 70×70×8 or about1×1×9, or about 5×5×9, or about 10×10×9, or about 15×15×9, or about20×20×9, or about 25×25×9, or about 30×30×9 or about 35×35×9 or about40×40×9 or about 45×45×9 or about 50×50×9 or about 55×55×9 or about60×60×9 or about 65×65×9 or about 70×70×9, or about 1×1×10, or about5×5×10, or about 10×10×10, or about 15×15×10, or about 20×20×10, orabout 25×25×10, or about 30×30×10 or about 35×35×10 or about 40×40×10 orabout 45×45×10 or about 50×50×10 or about 55×55×10 or about 60×60×10 orabout 65×65×10 or about 70×70×10 cubic microns. Preferably, the voxel is35×35×5 cubic microns.

A person skilled in the art will also appreciate that the voxel(s) ofthe reference standard and control sample(s) may be any dimensions. Asindicated above, the reference standard(s) and control sample(s) may bethe same volume as the test sample or they may be a different volume andthe skilled addressee will understand that calculations comparing thetest, reference standard(s) and control sample(s) must allow for anyvariations in volume between the test, reference standards and controlsamples. Accordingly, as described herein, the pre-defined voxelincludes, but is not limited to, a range of about 1 cubic micron toabout 8×10⁵ cubic microns. For example, the pre-defined voxel is in therange of about 1 cubic micron to 10,000 cubic microns, or about 10,000to 100,000 cubic microns, or about 100,000 to 800,000 cubic microns, Inone example, the pre-defined voxel is about 1 cubic micron to 10,000cubic microns, or about 2,000 cubic microns to about 8,000 cubicmicrons. In another example, the voxel is about 6,125 cubic microns.

The two-dimensional (2D) mapping between the pathological topography andthe atomic topography, reveals the relative abundance of manganese.

The method according to any aspect, embodiment or example of theinvention optionally includes a step of obtaining or deriving the testsample from a subject. For example, the sample is obtained from a“subject”, “participant”, or “patient” referred to herein as “subject”.The sample may be from a tissue or organ of the subject. The subject asdescribed according to any aspect, embodiment and/or example of theinvention includes, but is not limited to, any normal human or mammaliansubject or any human or mammalian subject in need of any treatment orprophylaxis as described according to the invention. The subjectincludes a subject diagnosed with a cancer, or any form of cancer orneoplasm described in accordance with the invention, or suspected ofhaving a cancer, or any form of cancer or neoplasm described inaccordance with the invention. It will also be understood by a personskilled in the art that the subject may be undergoing treatment and/orprophylaxis for any disorder including any cancer. The subject may beasymptomatic but has undergone a test or scan indicative of anunderlying condition, or may also be symptomatic. The subject includesthose undergoing clinical treatment including cancer therapy, orclinical intervention in the form of drugs, chemotherapy, immunotherapy,surgery, radiation or therapeutic devices, or those not yet undergoingany clinical treatment. Mammalian subjects include, but are not limitedto, apes, gorillas, chimpanzees, endangered species, stock animals,e.g., cattle, pigs, horses, and companion animals, e.g., dogs and cats.

The control sample according to the invention may be any suitable sampleand is preferably a biological sample. For example, the control samplemay comprise or be derived from a cell, a population of cells, one ormore single celled organism(s), a tissue sample or part thereof, anorgan sample or part thereof, one or more cells obtained/derived from aprokaryotic or eukaryotic organism, a population of cells and itsassociated non-cellular stromal components, a neoplastic cell orpopulation of neoplastic cells, a tissue sample from any organ or tissuefrom a subject, a tumour wherein the tumour for example, is a solid massor a “liquid” population of cells, for example, any cancer of thehematopoietic systems including leukemic cells, or the circulatingcellular derivatives of solid tumours, or derivatives of cells such asexosomes, including, but not limited to, a cell or population of cellsthat has metastasized. Preferably, the control sample comprises or isderived from a cell, a population of cells, a “normal” tissue sample, atissue sample of a tumour/neoplasm of cancers of the testes (e.g.,seminoma), lymphoma (e.g., B-cell lymphomas), small cell lung cancers,cancers of the brain (e.g., glioblastoma multiforme/astrocytoma),mesotheliomas, melanomas, and cancers of the breast and prostate.

In another aspect the present invention provides a method of identifyinga cancer that is likely to reoccur post radiation treatment comprising:

-   -   (a) quantifying level of manganese in a 3D region of a test        sample from the tumour by selecting a 2D region of said test        sample, wherein the 2D region is topographically defined by an        X′:Y′ coordinate system, wherein the 3D region corresponds to        said 2D region and has a selected height represented by Z,        wherein the 3D region is divided into three or more voxels of a        pre-defined volume, the volume of each voxel being defined by        X×Y×Z;    -   (b) measuring the central tendency level of manganese in each        voxel;    -   (c) identifying in the 2D region corresponding to the X and Y        coordinates of the voxel, high metallomic regions (HMRs), being        regions in which the level of manganese is higher than in the        surrounding areas as enabled by statistical thresholds that are        multiples of a central tendency or any approximation between        integers;        wherein when the frequency of HMRs is high it is indicative of        the likelihood of reoccurrence of the cancer post-radiation        treatment, and wherein when the frequency of HMRs is low, it is        indicative of the likelihood of non-reoccurrence of the cancer        post-radiation.

In another aspect the present invention provides a method according tothe invention, wherein the HMRs are also identified in the corresponding2D region of the test sample stained with a stain that distinguishescancer cells from others, preferably hematoxylin and eosin (H&E) stain.

In a further aspect, the present invention provides a method ofdetermining the radio-responsiveness of a tumour, the method comprisingdetermining the level of melanin in a test sample from the tumour,wherein the lower the level of melanin the more sensitive the tumour isto radiation and the higher the level of melanin the more resistant thetumour is to radiation.

The method or use according to any aspect, embodiment or example of theinvention optionally includes a step of obtaining or deriving thecontrol sample from a subject. For example, the control sample isobtained from a tissue or organ of a “subject”, “participant”, or“patient” referred to herein as “subject”. The subject as describedaccording to any aspect, embodiment and/or example of the inventionincludes, but is not limited to, any normal human or mammalian subjector any human or mammalian subject in need of any treatment orprophylaxis as described according to the invention. The subjectincludes a subject diagnosed with a cancer, or any form of cancer orneoplasm/tumour described in accordance with the invention, or suspectedof having a cancer, or any form of cancer or neoplasm/tumour describedin accordance with the invention. It will also be understood by a personskilled in the art that the subject may be undergoing treatment and/orprophylaxis for any disorder including any cancer. The subject may beasymptomatic but has undergone a test or scan indicative of anunderlying condition, or may also be symptomatic. The subject includesthose undergoing clinical treatment including cancer therapy, orclinical intervention in the form of drugs, chemotherapy, immunotherapy,surgery, radiation or therapeutic devices, or those not yet undergoingany clinical treatment. Mammalian subjects include, but are not limitedto, apes, gorillas, chimpanzees, endangered species, stock animals,e.g., cattle, pigs, horses, and companion animals, e.g., dogs and cats.The control sample may also be obtained from other species including,but not limited to, avian species such as a chicken, duck or goose.

It will be understood by the person skilled in the art that all types ofradiation are contemplated in any aspect, embodiment or exampledescribed herein for example, photon, or proton based, gamma rays, alpharays, beta rays. Any type of radiation that ionizes water irrespectiveof any other primary effects. For example, ¹³¹iodine for internallyirradiating rare tumours of the thyroid, or brachytherapy via insertedseeds for prostate cancer using radioactive ¹⁹³palladium or ¹²⁵iodine,all lead to radiolysis of water via low energy X-rays, or ²²³Radiumdichloride for metastases to lesions of bone which irradiate via alphaparticles. Similarly high dose brachytherapy which involves thetemporary insertion of needles containing ¹⁹²iridium, operates viaexactly the same mechanism. Without being bound to any particulartheory, whilst focus in the art has been on DNA repair, it is thoughtthat protein damage may be the issue and DNA problems may be secondary.Various damaging ions cause major cellular problems via damage toproteins.

Common measures of central tendency are the median, arithmetic mean andmode. Any other measures of central tendency known in the art may alsobe used, including but not limited to geometric mean, medimean,winsorized k-times mean, K-times trimmed mean and weighted mean, and thedata may also be transformed prior to calculating a central tendency.

Until the present invention, the clinical importance of manganese hadnot been recognised and manganese had not been quantified in order todetermine the radio-sensitivity/radio-resistance of a test sample inorder to make clinical decisions regarding radiation treatment.

It will be understood that “radiation sensitivity” or “sensitive toradiation” as used herein means that cells are either killed or aredisabled such that they do not divide further when exposed to radiation.“Radiation resistant” or “resistant to radiation” as used herein meansthat one or more cells remains viable after radiation treatment andis/are still able to divide allowing surviving tumour and stromal cellsto repopulate the irradiated site—this would be indicative that, iftreated with radiation, the primary tumour, or its metastaticderivatives in the subject, is likely to remain capable of growth and/orfurther metastasis.

It will be understood that the level of manganese in the test sample maybe compared to the level of manganese in one or more referencestandards(s) to calculate the ATI according to the invention.

The threshold ATI for use according to the invention will be apparent tothe person skilled in the art selecting the test sample, referencestandard(s) and/or controls for analysis. It will be understood that theATI threshold or thresholds may be based on known or derived CC/S levelsfor radio-responsiveness derived from a control sample or sampleswherein the control sample or samples comprise cells of the same cancertype as that of the test sample. For example, as shown by exampleherein, a radio-sensitivity threshold may be set at 2K calibrated countsper second (CC/S) for ⁵⁵Mn, which applies to a 35×35×5 cubic micronvoxel. The same numerical threshold may be set at 8K CC/S based on a70×70×5 cubic micron voxel. The threshold may also be set, for example,at 1 standard deviation above the central tendency value (preferably themean or median) obtained with the radiation sensitive control samples,or 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9 or 4.0 standard deviations, or any approximation between integers. Athreshold may also be set by empirical determination from differenttumour types. As shown by example herein, using melanomas as an example,the threshold may be set at a factor of 1.0×, 1.1×, 1.2×, 1.3×, 1.4×,1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2.0×, 2.1×, 2.2×, 2.3×, 2.4× or 2.5× abovethe central tendency value (preferably the median or mean), or anyapproximation between integers. A person skilled in the art willappreciate that the threshold will, in most cases, include allowing forthe “background” values in a given sample. For example, any test sampleATI that falls below the pre-determined ATI threshold set by referenceto the ATI of the radiation sensitive control, is considered to be anindication that the source of the test sample is sensitive to radiation;and any test sample ATI that falls above the pre-determined ATIthreshold set by reference to the ATI of the radiation resistantcontrol, is considered to be an indication that the source of the testsample is resistant to radiation.

As a further example, one pre-determined ATI threshold that can be usedto measure radio-responsiveness is 2K CC/S, since 87% of the values inFIG. 11 for the three radio-sensitive tumour types (seminoma, lymphomaand small cell lung) fall below 2K. For the two tumour types that areclinically agreed to be radio-resistant, namely mesothelioma and brain,86% of the values in FIG. 11 fall above 2K CC/S. However, the skilledaddressee will understand that a 2K threshold, while appropriate in somecircumstances, may not be in others as there will be variation betweenpatients depending on other factors, such as their genetic background.Thus a radio-sensitive threshold could be set, in some circumstances, at3K. The accepted clinical reality in oncology is that there are usuallytwo thresholds for any given outcome. Below the first threshold there isone confident outcome, above the second threshold there is a differentconfident outcome, and between the two thresholds there is an“intermediate zone” that is clinically heterogeneous and where theoutcome is varied. In the case of the data in FIG. 11, one could set thetwo thresholds at 3K and 4K, with the intermediate zone being 3K-4K.These thresholds will become clearer as more metallomic andradiotherapeutic data become available.

It will be clear to the skilled addressee that the invention can also beused to identify voxels within the 3D regions of the invention that haveparticularly high levels of metals, including manganese. For example, itis also contemplated that by setting the threshold for a voxel to appearas positive for manganese at a relatively high level, regions of highmetallomic content can be identified. As used herein, when referring toa specific metal in a High Metallomic Region (HMR), the HMR isdesignated as e.g., HMR(⁵⁵Mn), HMR(⁶⁶Zn), HMR(⁵⁶Fe) and HMR(⁶³Cu)respectively, with HMR(^(A)M) referring to the generic case of AnyMetal. It will be understood by the skilled addressee that the HMR forany metal (HMR(^(A)M) will minimally contain two adjacent voxels tofulfill the criterion of voxel contiguity. The size of HMR(^(A)M) canvary from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 etc contiguous voxels, toan integer which is the sample size chosen for analysis of thatparticular tumour.

A person skilled in the art will appreciate that the size of HMR(^(A)M)according to the invention is in any range measurable using existingtechnology. It will be appreciated that the maximum number that can beanalysed if a conventional slide is loaded to the edges with tumourmaterial will depend on the voxel size chosen. For example, HMR(^(A)M)size can range from 2 voxels to about 800,000 voxels and any value inbetween based upon a 35×35 (length by breadth) square micron size. Byway of illustration only, an empirically chosen 8×8 voxels (35×35 squaremicrons in 2D) efficiently revealed regions of high metallomic content,as shown in the examples. A minimal 8×8 voxel HMR-containing landscapeis bounded in both the X′ and Y′ directions. Thus HMRs above X8×Y8voxels can individually increase by integers in the X′ and Y′ 2Ddirections, yielding HMRs ofX[8+1]×Y[8]:X[8]×Y[8+1]:X[8+1]×Y[8+1]:X[8+2]×Y[8]:X[8+2]×Y[8+1]:X[8+2]×Y[8+2]:X[8+1]×Y[8+2]:X[8]×Y[8+2] through X[8+n]×Y[8+n] where n is an integer that can vary from 1to thousands, but preferably from 1 to 22, when a sample of 30×30 voxels(in 2D representing 1 mm²) is sampled. It will be appreciated that thethreshold may be set lower than a minimum of 8 voxels. The value of 8has been used here because it is an empirically-derived efficient searchtool for HMRs.

A person skilled in the art will also appreciate that an HMR can applyto cancer cells in a tumour, or to a region of cellular and non-cellularmaterial, generally referred to as associated stroma, which itself canexist in an “activated” state owing to its interaction with neighbouringcancer cells.

The amount of radiation and/or anti-cancer therapy actually administeredaccording to the invention will typically be determined by a physicianin the light of the relevant circumstances, including the condition tobe treated, in view of other options such as chemotherapy andimmunotherapies, the age, weight, and response of the individualpatient, the severity of the patient's symptoms/condition, and the like.The radiation therapy includes, but is not limited to, stereotactic bodyradiation therapy, fractionated external beam radiation therapy andbrachytherapy, the external beam radiation therapies (EBRT) includeintensity-modulated radiotherapy, conformal radiation, stereotactic bodyradiation, proton beam radiation, GammaKnife and CyberKnife, andbrachytherapy includes permanent implantation of radioactive isotopic“seeds” as well as temporary high dose radioactivity seeds. The type ofradiation delivered to a patient may be photon or proton based, and maybe delivered via a linear accelerator, gamma rays (from any source), orvia radioactive seeds comprising ¹²⁹Iodine, ¹³¹Cesium and ¹³³Cesium or¹⁰³Palladium, for LDRBT, or ¹⁹²Iridium (for High Dose Rate Brachytherapy(HDRBT), or ⁹⁰Yttrium resin microspheres or ³²P conjugated to siliconmicroparticles. The radiation can also be delivered via other entitiesset out below (Chellan and Sadler, 2015, Phil. Trans. R. Soc, A.373:20140182)—for example, ⁹Be bound to protein as a neoantigen stimulant ofthe immune response for immunotherapies combined with radiation;⁸⁹Strontium for osteoblastic bone metastases; ²²³Radium for treatment ofbone metastases and castration-resistant prostate cancer; ⁴⁷Scandium and⁴⁴Scandium as therapeutic radionuclides; ⁵⁹Nickel bound to the surfaceof the MHC and peptides for triggering the immune response of T cells;⁹⁰Yttrium targeted to somatostatin receptors for cancer treatment;⁹⁶Molybdenum as Molybdate as a preventative antioxidant of lipids andthe treatment of breast and oesophageal cancer; ¹⁰¹Ruthenium delivery tocancer cells via serum transferrin; ¹⁰⁵Rhodium for bone metastases;¹⁰³Palladium as brachytherapy for prostate cancer and choroidalmelanoma; ¹⁷⁸Hafnium as nanospheres for efficient uptake by tumour cellsand its enhancement of radiation effects for soft tissue sarcomas andhead and neck cancer; ¹⁸⁴Tungsten as polyoxotungstates as anticanceragents; ¹⁸⁸Rhenium and ¹⁸⁶Rhenium for small cell lung cancer andprostate cancer; ¹⁹⁰Osmium as a superoxide mimic and organo-osmium arenecomplexes as anticancer drugs; ¹⁹⁵Platinum in cancer chemotherapy;¹⁵³Samarium for osteosarcoma and metastatic breast cancer to bones;¹⁶⁶Holmium for internal radiation therapy; ¹⁷⁵Ytterbium labelledpolyaminophosphonates for bone metastases; ¹⁷⁷Lutetium labelled peptidesand antibodies for small cell lung cancer; ²²⁵Actinium for myeloidcancers and its decay product ²¹³Bismuth; ²⁸Silicon containingphthalocyanine as a photosensitizer for killing cancer cells; ²¹²Leadgenerating ²¹²Bi for radioimmunotherapy in combination with trastuzumabfor binding to HER2 and delivering radiation upon internalization ofdifferent cancer cell types; ³²P as phosphocol to treat differentcancers; ⁷⁵Arsenic as As₂O₃ for promyelocytic leukemia, unresectablehepatocellular carcinoma and non-small-cell lung cancer; ²¹³Bismuthlabelled lintuzumab for targeted radiotherapy of Acute Myeloid leukemia;⁷⁹Selenium for chemoprotection of prostate cancer; ¹²⁷Iodine and¹³¹Iodine for thyroid cancer; and ²¹¹Astatine for elimination of tumorcells in the brain and in recurrent ovarian cancer. In another example,the substance includes a radiosensitizer, such as, but not limited toboron (¹⁰B), Rose Bengal, 2-deoxy-D-glucose, or immunotherapeuticadditions that are combined with radiation (Sharabi et al., Oncology[Williston Park] 2015, 29(5), pii:211304; Formenti, J Natl Cancer Inst105, 256-265, 2013).

In the context of the present invention, the letters X′, Y′ and Z in theterm “X′×Y′×Z” refer to the dimensions of a “3D region” of the sampleand “X′×Y′×Z” relates to length×breadth×height of the 3D region whichprovides a volume of the 3D region.

In the context of the present invention, the letters X, Y and Z in theterm “X×Y×Z” refer to the dimensions of a voxel within a “3D region” ofthe sample and “X×Y×Z” relates to length×breadth×height of the voxelwhich provides a volume of the voxel.

Accordingly, in the context of the present invention, where terms suchas “35×35×5” appear, they relate to a length×breadth×height and providea measure of volume; whereas when terms “35×35” appear they relate tolength×breadth and provide an area (2D).

In another aspect, the present invention provides a method ofdetermining radio-responsiveness of a melanoma, the method comprisingdetermining the level of melanin in a test sample from the melanoma,wherein the lower the level of melanin in the test sample the moresensitive the melanoma is to radiation and the higher the level ofmelanin in the test sample the more resistant the melanoma is toradiation.

In another aspect, the present invention provides a method ofdetermining radio-responsiveness of a melanoma, the method comprisingcomparing the level of melanin in a test sample from the melanoma to apre-determined melanin threshold wherein the radio-responsiveness of themelanoma is determined by assessing whether the level of melanin in thetest sample is above or below the melanin threshold,

-   -   wherein if the level of melanin in the test sample is below the        melanin threshold the melanoma is determined to be sensitive to        radiation; and    -   wherein if the level of melanin in the test sample is above the        melanin threshold the melanoma is determined to be resistant to        radiation.

In one or more embodiments, the level of melanin in the test sample iscompared to two pre-determined melanin thresholds wherein theradio-responsiveness of the melanoma is determined by assessing whetherthe level of melanin in the test sample is above or below the twothresholds, and

-   -   wherein if the melanin in the test sample is below the lower        melanin threshold the melanoma is determined to be sensitive to        radiation;    -   wherein if the melanin in the test sample is above the higher        melanin threshold the melanoma is determined to be resistant to        radiation; and    -   wherein if the melanin in the test sample is between the two        melanin thresholds, the melanoma is determined to be partially        sensitive to radiation.

It is well within the competence of the skilled addressee to set thepre-determined melanin threshold. Voxels containing melaninconcentrations in a section can first be determined using metal-labelledmelanin antibodies to the section that is ablated via any form ofelemental analysis (such as LA-ICP-MS), while simultaneously measuring⁵⁵Mn levels in the same voxels. Voxels that contain a level of melaninthat exceeds a threshold level of the same metal labelled antibodyapplied to an amelanotic melanoma sample, are informative. Thepercentage of melanotic voxels within a test sample, together with aweighted median of those voxels provides a radio-protective index. Theskilled addressee will note that thresholds for melanin can be set inthe same general manner as for the ATI in terms of a central tendency,preferably the median or mean, in appropriate units. It should also benoted that ⁵⁵Mn may be measured simultaneously in the same voxels asthose analyzed for melanin.

For example, the threshold could be set at the mean or median of melaninlevels in melanomas from a cohort that has responded differently toradiation treatment (i.e. some were sensitive and some resistant).Alternatively, two thresholds could be set in which case, as an example,they could be set at one or at two standard deviations from the mean ofmelanin levels in a set of melanomas that have responded differently toradiation treatment eg a cohort in which there is an equal number ofradiation resistant and radiation sensitive melanomas.

In another aspect, the present invention provides a method of treatingmelanoma in a subject comprising performing the method of the inventionon a test sample from the subject, and including radiotherapy intreating said melanoma in the subject if the melanoma is determined tobe sensitive to radiation.

In another aspect, the present invention provides a method of treatingmelanoma in a subject comprising, performing the method of the inventionon a test sample from the subject, and not including radiotherapy intreating said melanoma in the subject if the melanoma is determined tobe resistant to radiation.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

Each aspect, embodiment and/or example of the invention described hereinis to be applied mutatis mutandis to each and every aspect, embodimentand/or example unless specifically stated otherwise.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

It will be acknowledged by the skilled addressee that the methods of thepresent invention are not in any way routine or conventional in aclinical therapeutic content. The precision that the present inventionprovides currently does not exist. Distinguishing tumour types (inparticular the eight tumour types exemplified) on the basis of, forexample, LA-ICP-MS is entirely new; the results are unexpected and couldnot have been predicted from the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Photographic image of a standard 5 micron, H&E stained tissuesection from a formalin-fixed paraffin-embedded (FFPE) block from astage Ib malignant melanoma of the neck of a 48 year old male, with theTNM staging criteria of T2aN0M0, where T2 represents invasion of themuscularis propria; N0 represents no lymph node metastasis, and M0represents no distant metastasis. Note that the top right-hand side ofthis section consists of pale amelanotic tumour cells, while the darkerregions on the left-hand side and bottom of the Figure are composed ofcells containing darkly staining melanin, and with even larger patchesof melanin, both intra- and extra-cellular, as black spots in the lowerpart of the image.

FIG. 2: Illustration of three different manganese-based bindingentities. It should be noted that a single ⁵⁵Mn²⁺ does not bind allthree entities at once (this figure is for illustrative purposesonly—taken from Slade and Radman, Microbiology and Molecular BiologyReviews, 75, 133-191, 2011.)

FIG. 3: A. Photographic image of an H&E stained 5 micron tissue sectionfrom a block of a normal human cortex from a 50 year old human femaleshowing the characteristics of normal cortical tissue. It isillustrative of the position of a three track ablation (taken from thelarger multi-track tissue ablation) that was carried out on an adjacentunstained section from the same block, but on a different slide. The H&Estained area shown in the Figure corresponds to the equivalent unstainedarea analysed by LA-ICP-MS on a different slide. B. The calibratedsignal of each of three metals ⁵⁵Mn, ⁶⁶Zn, and ⁵⁶Fe are shown for eachvoxel of three contiguous ablation tracks from a tissue section thatablated 70 voxels per track. C. Graphical representation of the rawnumerical values (from FIG. 3B), wherein each track consisted of 70voxels of calibrated counts per second for the three metals, ⁵⁵Mn, ⁶⁶Znand ⁵⁶Fe in adjacent voxels in each of three ablation rows.

FIG. 4: A. Photographic image of an H&E stained 5 micron tissue sectionfrom a block of a 19 year old human female with a brain neoplasmclassified as glioblastoma multiforme. The H&E stained area shown in theFigure corresponds to the equivalent unstained area analysed byLA-ICP-MS on a different slide. B. The calibrated signal of each ofthree metals ⁵⁵Mn, ⁶⁶Zn, and ⁵⁶Fe are shown for each voxel of threecontiguous ablation tracks from a tissue section that ablated 68 voxelsper track. C. Graphical representation of the raw numerical values (fromFIG. 4B) of calibrated counts per second for the three metals, ⁵⁵Mn,⁶⁶Zn and ⁵⁶Fe in adjacent voxels in each of three ablation rows from thehuman female with glioblastoma multiforme. The Y axis shows calibratedcounts per second (CC/S) of that metal ion, while the X axis shows thevoxel number of the three ablation tracks.

FIG. 5: A. Photographic image of an H&E stained 5 micron tissue sectionfrom a different region of the same block of the 19 year old humanfemale with glioblastoma multiforme shown in FIG. 4. B. The calibratedsignal of each of three metals ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe are shown for eachvoxel of three contiguous ablation tracks from a tissue section thatablated 69 voxels per track. C. Graphical representation of rawnumerical values (from FIG. 5B) of calibrated counts per second for thethree metals, ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe in adjacent voxels in each of threeablation rows from the human female with glioblastoma multiforme. The Yaxis shows calibrated counts per second (CC/S) of that metal ion, whilethe X axis shows the voxel number of the three ablation tracks.

FIG. 6: A. Photographic image of an H&E stained 5 micron tissue sectionfrom a 60 year old human male with malignant mesothelioma. B. Thecalibrated signal of each of four metals ⁵⁵Mn, ⁶⁶Zn, ⁵⁶Fe and ⁶³Cu areshown for each voxel of three contiguous ablation tracks from a tissuesection that ablated 67 voxels per track. C. Graphical representation ofthe raw numerical values (from FIG. 6B) of calibrated counts per secondfor the four metals, ⁵⁵Mn, ⁶⁶Zn, ⁵⁶Fe and ⁶³Cu in adjacent voxels ineach of three ablation rows from the 60 year old human male withmalignant mesothelioma. The Y axis shows calibrated counts per second(CC/S) of that metal ion, while the X axis shows the voxel number of thethree ablation tracks.

FIG. 7: A. Photographic image of an H&E stained 5 micron tissue sectionfrom a 50 year old male with malignant melanoma of the esophagus. B. Thecalibrated signal of each of three metals ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe are shownfor each voxel of three contiguous ablation tracks from a tissue sectionthat ablated 68 voxels per track. C. Graphical representation of the rawnumerical values (from FIG. 7B) of calibrated counts per second for thethree metals, ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe in adjacent voxels in each of threeablation rows from the 50 year old human male with malignant melanoma ofthe esophagus. The Y axis shows calibrated counts per second (CC/S) ofthat metal ion, while the X axis shows the voxel number of the threeablation tracks.

FIG. 8: A. Photographic image of an H&E stained 5 micron tissue sectionfrom a 57 year old male with diffuse B-cell lymphoma. B. The calibratedsignal of each of three metals ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe are shown for eachvoxel of three contiguous ablation tracks from a tissue section thatablated 79 voxels per track. C. Graphical representation of the rawnumerical values (from FIG. 8B) of calibrated counts per second for thethree metals, ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe in adjacent voxels in each of threeablation rows from the 57 year old male with diffuse B-cell lymphoma inthe testis. The Y axis shows calibrated counts per second (CC/S) of thatmetal ion, while the X axis shows the voxel number of the three ablationtracks.

FIG. 9: A. Photographic image of a representative H&E stained 5 microntissue section from a 38 year old male with a small cellundifferentiated malignant carcinoma of the lung. B. The calibratedsignal of each of three metals ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe are shown for eachvoxel of three contiguous ablation tracks from a tissue section thatablated 65 voxels per track. C. Graphical representation of the rawnumerical values (from FIG. 9B) of calibrated counts per second for thethree metals, ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe in adjacent voxels in each of threeablation rows from the 38 year old male with a small cellundifferentiated malignant carcinoma of the lung. The Y axis showscalibrated counts per second (CC/S) of that metal ion, while the X axisshows the voxel number of the three ablation tracks.

FIG. 10: Photographic image of a representative H&E stained 5 microntissue section from a 52 year old male with seminoma. B. The calibratedsignal of each of three metals ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe are shown for eachvoxel of three contiguous ablation tracks from a tissue section thatablated 79 voxels per track. C. Graphical representation of the rawnumerical values (from FIG. 10B) of calibrated counts per second for thethree metals, ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe in adjacent voxels in each of threeablation rows from the 52 year old male with seminoma. The Y axis showscalibrated counts per second (CC/S) of that metal ion, while the X axisshows the voxel number of the three ablation tracks.

FIG. 11: Graphical representation of total median manganese contents,(expressed as calibrated counts per second, CC/S of ⁵⁵Mn), in tumoursfrom patients with seminoma (55), lymphoma (10), small cell carcinoma ofthe lung (20), melanoma (64), brain (glioblastoma and astrocytoma) (25),and mesothelioma (10). Each square represents a single patient except inthe melanomas, where two of the 64 patients each had two major lineageswithin their tumours, and each lineage was represented separately in thehistogram with a square.

FIG. 12: Two-dimensional representation of laser ablation tracks acrossthe top part of the tissue section shown in FIG. 1 of one of themelanoma patients whose tumour had two major lineages within it. Scale;right hand side, (dark grey), 0-15,000; middle, (whitish),15,000-45,000; left hand side, (pale grey), 45,000-150,000 calibratedcounts per second.

FIG. 13: Graphical representation of an analysis of all four metals,with panels (a), (b), (c) and (d) showing ⁵⁵Mn, ⁶⁶Zn, ⁵⁶Fe and ⁶³Curespectively of the tumour shown in FIG. 1.

FIG. 14: Photographic image of an H&E stained tissue section of amelanoma from the left forefinger of a 54 year old male: stage IV,T4N0M1 (upper panel). Two-dimensional representation of ablation tracksacross the tumour (lower panel). Scale; pale grey (main bulk ⁵⁵Mn valueof the tumour) 0-2,000 CC/S; whitish flat areas, generically termedherein as High Metallomic Regions (HMRs), which in this particularexample correspond to >6,500 calibrated counts per second.

FIG. 15: A. Photographic image of a standard 5 micron H&E stained tissuesection from a formalin-fixed paraffin-embedded block from a melanoma onthe left forefinger of the 54 year old male analyzed in FIG. 14illustrating the tissue and cellular morphology. B. The metallomiccontent of the sample shown in A was measured. Part B is atwo-dimensional representation of ⁵⁵Mn levels in individual voxels ofthis section after LA-ICP-MS. Two clusters of contiguous voxels of high⁵⁵Mn content, denoted HMRs (⁵⁵Mn) and shown as black areas, becomevisible after visual inspection of the data of the voxel matrix. C.Two-dimensional representation of ⁵⁵Mn levels in individual voxels ofthe section of A and B were further analyzed by applying a threshold of2× the median value per voxel of the whole area, (plus the machinebackground value). Black squares show those voxels that are above thisparticular (2× median) threshold. D. Two dimensional representation of⁵⁵Mn levels in each individual voxels of the section of A and B werefurther analyzed by applying a threshold of the median value per voxelof the whole area, plus the standard deviation (St.Dev.) of the voxelvalues of the whole area, plus the machine background value. Blacksquares show those voxels that are above this particular (median+St.Dev)threshold.

FIG. 16: Top panel. Photographic image of the same standard 5 micron H&Estained tissue section from the tumour of the melanoma patient frompanel A of FIG. 15. Middle panel. Two-dimensional representation of thesame voxel matrix of ⁵⁵Mn values shown in panel C of FIG. 15 showing thetwo clusters of contiguous voxels that constitute the HMRs (⁵⁵Mn). Lowerpanel. ⁶⁶Zn voxel values in the identical voxels to those shown in themiddle panel and using the same threshold criterion (2× median) for ⁶⁶Znas for ⁵⁵Mn.

FIG. 17: Two-dimensional representation of the distribution andemergence of HMRs (⁵⁵Mn) in voxel matrices generated via LA-ICP-MS usingdifferent threshold criteria. The four left hand side panels (T1, T2, T3and T4) show data from laser ablation of a 31×31 voxel area from astandard unstained 5 micron tissue section of a small cellundifferentiated carcinoma of the lung from a 51 year old femalepatient. The four right hand side panels (T1, T2, T3 and T4) show datafrom laser ablation of a 31×31 voxel area of the 54 year old melanomapatient described in FIGS. 14 to 16. The ⁵⁵Mn threshold values appliedto the voxel matrix data are; T1, 0.5× median; T2, 1× median; T3, 1.5×median and T4, 2× median. Dark voxels in each panel are those in which⁵⁵Mn values exceed the threshold for that panel. Single non-contiguousvoxels above threshold are designated as singletons, while twocontiguous voxels are designated as doublets.

FIG. 18: Two-dimensional representation of the distribution and noemergence of HMRs(⁶⁶Zn) in voxel matrices generated via LA-ICP-MS forthe same samples from the two patients of FIG. 17. The four left handside panels (T1, T2, T3 and T4) show data from laser ablation of thesame 31×31 voxel area as in FIG. 17 which was simultaneously analyzedfor ⁶⁶Zn, ⁵⁶Fe and ⁶³Cu. The data for ⁶⁶Zn are shown. The four righthand side panels (T1, T2, T3 and T4) show data from laser ablation ofthe same 31×31 voxel area as in FIG. 17. The ⁶⁶Zn threshold valuesapplied to the voxel matrix data are as used previously; T1, 0.5×median; T2, 1× median; T3, 1.5× median and T4, 2× median. Dark voxels ineach panel are those in which ⁶⁶Zn values exceed the threshold for thatpanel.

FIG. 19: Histograms showing median ⁵⁵Mn contents expressed as CalibratedCounts per Second (CC/S) of ⁵⁵Mn in laser ablated samples from tumoursof 20 patients with small cell carcinomas of the lung. Each grey squarerepresents the median value calculated from a total area ofapproximately 1,800 voxels from the tumour of a single patient which wassampled. The top histogram illustrates 14 patients with tumours withoutHMRs(⁵⁵Mn). The lower histogram shows tumours with HMRs(⁵⁵Mn) from sixpatients denoted t1 through t6 which were determined at the standard T4threshold of 2× median. Black squares are the median values of theHMRs(⁵⁵Mn) found in the tumour from each of patients t1 to t6 determinedusing the standard T4 threshold of 2× median. The HMR(⁵⁵Mn) values arejoined via a dotted line to the bulk median ⁵⁵Mn value of that tumourfor each of patients t1 to t6. The bins in the histogram are 200 CC/Sunits.

FIG. 20: Schematic representation of the size, shape and content of aselected sample of contiguous voxel configurations in a simulated 2Dtumour landscape. Voxels containing cancerous cells above a designatedthreshold are shown in black. Panel A illustrates the position of alleight possible voxel configurations when only voxel doublets areexamined. Panels B and E illustrate one of the many possible contiguousvoxel configurations that satisfy the criterion of an 8×8 abovethreshold HMR(^(A)M), where ^(A)M represents Any Metal. Panel Cillustrates a voxel configuration that is only 7×7 voxels which wouldfall below an 8×8 minimal threshold. Panels D and F illustrate voxelconfigurations that would be characteristic of lymphatic vessels orducts. Panel G illustrates a voxel configuration that is characteristicof “single file” movements of cancerous cells, or of significant machine“stutter”. Panel H is characteristic of the multiple voxelconfigurations seen in some melanomas where the voxels above thresholdcan be due either to cells with high levels of any metal, or melaningranules that bind any metal.

FIG. 21: Histogram showing the median ⁵⁵Mn contents expressed asCalibrated Counts per Second (CC/S) of ⁵⁵Mn in laser ablated samplesfrom a total area of approximately 1800 voxels of tumours of 10 patientswith diffuse B cell lymphoma. Each grey square represents the medianvalue calculated from the tumour of a single patient. The bins in thehistogram are 200 CC/S units.

FIG. 22: Histograms of median ⁵⁵Mn contents expressed as CalibratedCounts per Second (CC/S) of ⁵⁵Mn in laser ablated samples from tumoursof 55 patients with classic seminoma. Each grey square represents themedian value calculated from a total area of approximately 1,800 voxelsof the tumour of a single patient. The top histogram illustrates the 51seminomas without HMRs(⁵⁵Mn). The lower histogram shows tumours withHMRs(⁵⁵Mn) from four patients denoted S4 through S7 which weredetermined at the standard T4 threshold of 2× median. Black squares arethe median values of the HMRs(⁵⁵Mn) found in the tumour from each ofpatients S4 to S7. The HMR(⁵⁵Mn) values are joined via a dotted line tothe bulk median ⁵⁵Mn value from the tumour of each of patients S4 to S7.The bins in the histogram are 200 CC/S units. Patients denoted 51, S2and S3 are outliers with high median values, even though their tumoursdo not contain HMRs(⁵⁵Mn) under the standard threshold.

FIG. 23: A histogram of median ⁵⁵Mn contents expressed as CalibratedCounts per Second (CC/S) of ⁵⁵Mn in laser ablated samples from tumoursof 10 patients with mesothelioma. Each grey square represents the medianvalue calculated from a total area of approximately 1,800 voxels of thetumour of a single patient. The top histogram illustrates 9 tumourswithout HMRs(⁵⁵Mn). The lower histogram shows a tumour with a singleHMRs(⁵⁵Mn) determined at the standard T4 threshold of 2× median. Theblack square is the median value of the HMR (⁵⁵Mn) found in the tumourof one mesothelioma patient. The HMR(⁵⁵Mn) value is joined via a dottedline to the bulk median ⁵⁵Mn value of that tumour. The bins in thehistogram are 200 CC/S units.

FIG. 24: Histograms showing median ⁵⁵Mn contents expressed as CalibratedCounts per Second (CC/S) of ⁵⁵Mn in laser ablated samples from tumoursof 25 patients with either glioblastoma multiforme or astrocytomas ofthe brain. Each grey square represents the median value calculated froma total area of approximately 1800 voxels of the tumour of a singlepatient. The top histogram illustrates 24 tumours without HMRs(⁵⁵Mn).The lower histogram shows a tumour with a single HMRs(⁵⁵Mn) determinedat the standard T4 threshold of 2× median. The black square is themedian value of the HMR (⁵⁵Mn) found in that tumour. The HMR(⁵⁵Mn) valueis joined via a dotted line to the bulk median ⁵⁵Mn value of thattumour. The bins in the histogram are 200 CC/S units.

FIG. 25: Histograms showing median ⁵⁵Mn contents expressed as CalibratedCounts per Second (CC/S) of ⁵⁵Mn in laser ablated samples from tumoursof 64 patients with melanoma. Each grey square represents the medianvalue calculated from a total area of approximately 1,800 voxels of thetumour of a single patient, except in the case of the two melanomapatients each with two major lineages within the same tumour, where thesampled area is less than 900 voxels per lineage. The top histogramillustrates the tumours without HMRs(⁵⁵Mn). The lower histogram showsthe tumours with HMRs(⁵⁵Mn) determined at the standard T4 threshold of2× median. Black squares are the median values of the HMRs(⁵⁵Mn) foundin the tumours. The HMR⁵⁵Mn) values are joined via a dotted line to thebulk median ⁵⁵Mn value of that tumour. Some tumours have multipleHMRs(⁵⁵Mn) and these are shown on the same dotted line for that tumour.The bins in the histogram are 200 CC/S units. Note that the scale hasbeen compressed since many CC/S values exceed values of 10,000.

FIG. 26: Schematic diagram of the complex polymeric structure of melaninwhich binds the metals used for analysis in this application.

FIG. 27: Panels A and B are photographic images of a standard 5 micronH&E stained tissue section from a formalin-fixed paraffin-embedded blockfrom a malignant melanoma of the chest wall of a 45 year old femaleillustrating the tissue and cellular morphology. C. ⁶⁶Zn levels inindividual voxels of this section with contiguous voxels of high ⁶⁶Zncontent shown as black areas. D. ⁶³Cu levels in individual voxels ofthis section with contiguous voxels of high ⁶³Cu content shown as blackareas. E. ⁵⁶Fe levels in individual voxels of this section withcontiguous voxels of high ⁵⁶Fe content shown as black areas. F. ⁵⁵Mnlevels in individual voxels of this section with contiguous voxels ofhigh ⁵⁵Mn content shown as black areas.

FIG. 28: Histograms showing median ⁵⁵Mn contents expressed as CalibratedCounts per Second (CC/S) of ⁵⁵Mn in laser ablated samples from a totalarea of approximately 1,800 voxels of the tumours of 64 patients withmelanoma. As described previously, two of the 64 patients each had twomajor lineages within their tumours, and each such lineage isrepresented separately in the histogram with a square. The melanomasamples have been grouped into those that derive from the primary site(top histogram), those that derive from lymph nodes, (middle histogram),and those that derive from a distant site (lower histogram).

FIG. 29: A. Photographic image of a standard 5 micron H&E stained tissuesection from a formalin-fixed paraffin-embedded block from a small cellcarcinoma of the lung of a 54 year old male illustrating the tissue andcellular morphology of areas of darkly staining cancerous cells and themore lightly staining stromal regions. B. 2D relief image of the samesection with ⁶⁶Zn levels above a selected threshold shown in white/greyand the stromal components below this threshold shown in black.

FIG. 30: A. Photographic image of a standard 5 micron H&E stained tissuesection from a formalin-fixed paraffin-embedded block from a malignantmelanoma of the rectum of a 66 year old male illustrating the tissue andcellular morphology of areas of darkly staining cancerous cells, evendarker staining melanin concentrations, and the more lightly stainingstromal regions. B. 2D relief image of the same section with ⁶⁶Zn levelsabove a selected threshold shown in white/grey and the stromalcomponents below this threshold shown in black.

FIG. 31: Histograms of median ⁵⁵Mn contents expressed as CalibratedCounts per Second (CC/S) of ⁵⁵Mn in laser ablated samples from tumoursof 15 patients with breast cancer. Each grey square represents themedian value calculated from a total area of approximately 1,800 voxelsof the tumour of a single patient. The top histogram illustrates 4tumours without HMRs(⁵⁵Mn). The lower histogram shows 11 tumours withHMRs(⁵⁵Mn) determined at the standard T4 threshold of 2× median. Blacksquares are the median values of the HMRs(⁵⁵Mn) found in the tumour ofeach of the 11 patients. The HMR⁵⁵Mn) values are joined via a dottedline to the bulk median ⁵⁵Mn value of that tumour. Some tumours havemultiple HMRs(⁵⁵Mn) and these are shown on the same dotted line for thattumour. The bins in the histogram are 200 CC/S units.

FIG. 32: A. Photographic image of a standard 5 micron H&E stained tissuesection from a formalin-fixed paraffin-embedded block from welldifferentiated carcinoma of the breast taken from a 39 old femaleillustrating the tissue and cellular morphology. B. 2D relief image of⁵⁵Mn levels in individual voxels of this section after LA-ICP-MS.Multiple clusters of contiguous voxels of high ⁵⁵Mn content are shown asblack areas.

FIG. 33: Two-dimensional representation of distribution and emergence ofHMRs(⁵⁵Mn), but no emergence of HMRs(⁶⁶Zn) in voxel matrices generatedvia LA-ICP-MS using different threshold criteria from the 39 year oldfemale in FIG. 32. The four left hand side panels (T1, T2, T3 and T4)are ⁵⁵Mn data from laser ablation of a 31×31 voxel area from carcinomaof the breast at the different standard thresholds, (T1, 0.5× median;T2, 1× median; T3, 1.5× median and T4, 2× median). The four right handside panels (T1, T2, T3 and T4) are ⁶⁶Zn data from laser ablation of theidentical 31×31 voxel area and the same threshold criteria. Dark voxelsin each panel are those in which ⁵⁵Mn or ⁶⁶Zn values exceeded thethreshold for that panel.

FIG. 34: Analysis of the variation in the distribution of ⁵⁵Mn and ⁶⁶Znvoxel values in the breast cancer sample from the 39 year old femalewhose 2D data were presented in FIG. 33. Panel A shows the right handside Skew of the ⁵⁵Mn voxels, with the frequency of voxels in aparticular bin on the Y axis, and voxel values on the X axis. Panel Billustrates the near symmetrical distribution of ⁶⁶Zn voxel values fromthe same sample.

FIG. 35: A. Photographic image of a standard 5 micron H&E stained tissuesection from a formalin-fixed paraffin-embedded block from an invasiveductal carcinoma of the breast from a 48 year old female illustratingthe heterogeneous tissue and cellular morphology. B. Description of themajor morphological features of the above image, showing cancerous cellsin lymphatic ducts, (C1 through C5), normal ducts (N) surrounded byassociated adipocytes, and a concentration of immune cells, (immune),together with other stromal components.

FIG. 36: Top two panels are photographic images of a standard 5 micronH&E stained tissue section from a formalin-fixed paraffin-embedded blockfrom an invasive ductal carcinoma of the breast from the 48 year oldfemale of FIG. 35, illustrating the heterogeneous tissue and cellularmorphology. Bottom four panels. 2D relief images of the differentialdistribution of the four metals ⁵⁵Mn, ⁶⁶Zn, ⁵⁶Fe and ⁶³Cu in the areascontaining adipocytes and in some parts of the stromal regions.

FIG. 37: A. Illustration of the clinical data from patient X withadenocarcinoma of the prostate and a PSA of 8.1 showing theabnormalities in different regions of the prostate with 7 out of 12regions showing changes of little significance, and five regions with“cancer” as defined by Gleason scores no higher than 7 (upper panel). B.Diagnostic summary (Part A through Part L) of the extent of involvementof the twelve core needle biopsies from patient X.

FIG. 38: Histograms of median ⁵⁵Mn contents expressed as CalibratedCounts per Second (CC/S) of ⁵⁵Mn in laser ablated samples from tumoursof 10 patients with cancer of the prostate. Each grey square representsthe median value calculated from a total area of approximately 1800voxels of the tumour of a single patient. No HMRs(⁵⁵Mn) were presentunder the standard threshold conditions of 2× median applied herein.

FIG. 39 Magnetic Resonance Images from the brain of patient Y, a 70 yearold male. A. Presence of a contrast enhancing lesion in the paramedialleft parietal lobe of the brain (arrowed) prior to drug treatment andradiation. B. MRI of the same brain region after radiation andimmunotherapeutic drug treatment. C. Photographic image of a standard 5micron H&E stained tissue section from a formalin-fixedparaffin-embedded block from a primary melanoma of the same patientresected years prior to the brain scans. D and E. Higher powerillustrations of the regions of interest analyzed by LA-ICP-MS.

FIG. 40: The calibrated signal tracks (arrowed →) of ⁵⁵Mn from fiveseparate areas of cancerous cells of the primary melanoma of patient Y.There were six contiguous ablation tracks of standard 35 micron×35micron×5 micron voxels in each chosen area, with track lengths of 19,19, 13, 19 and 19 voxels respectively. The raw numerical values ofcalibrated counts per second for ⁵⁵Mn are shown for each of the adjacentvoxels in each of six ablation rows.

FIG. 41: Top panel. Patient Z with a squamous carcinoma of the oralregion prior to radiation treatment. Bottom panel. Patient Z six monthsafter radiation treatment.

FIG. 42: Flow diagram for melanoma patients whose solid tumour(s) areanalyzed by LA-ICP-MS. The optional pathways that provide evidence for aphysician as to whether to advise radiation treatment for the patient,or to refrain from the use of such a modality, are based on thehomogeneity or heterogeneity of the ATI. The key decision points concernwhether the ⁵⁵Mn voxel values fall above or below certain designatedthresholds, and the degree of melanization of the tumour, in terms ofmetal binding.

FIG. 43: Flow diagram for the non-melanoma patients whose solidtumour(s) are analyzed by LA-ICP-MS. The optional pathways that provideevidence for a physician as to whether to advise radiation treatment forthe patient, or to refrain from the use of such a modality, are based onthe homogeneity or heterogeneity of the ATI. The key decision pointsconcern whether the ⁵⁵Mn voxel values fall above or below certaindesignated thresholds.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In work leading up to the present invention the inventor noted that the2D spatial characteristics of many tumours, in particular, melanoma,varies greatly, e.g., as shown in FIG. 1 for comparative purposes. FIG.1 shows a standard 5 micron, H&E stained tissue section from aformalin-fixed paraffin-embedded (FFPE) block from a stage Ib malignantmelanoma of the neck of a 48 year old male, with the TNM stagingcriteria of T2aN0M0, where T2 represents invasion of the muscularispropria; N0 represents no lymph node metastasis, and M0 represents nodistant metastasis. Note that the top right-hand side of this sectionconsists of pale amelanotic tumour cells, while the darker regions onthe left-hand side and bottom of the Figure are composed of cellscontaining darkly staining melanin, and with even larger patches ofmelanin, both intra- and extra-cellular, as black spots in the lowerpart of the image. As described above herein, the pathological diagnosisof such tumours, which is utilized to determine treatment modalities,and upon which the decision is made as to whether or not radiation willbe used, is the subjective analysis of such H&E stained slides. Whilstthe pathological diagnosis is also aided by probing such tissue sectionswith a panel of antibodies that vary from one type of tumour type to thenext, such results are also subjective and far from quantitative. Untilthe present invention, there was no quantitative test available, or atest that predicts sensitivity or resistance to radiation, that aids theoncologist in making a decision on whether or not to utilize radiationas part of a treatment regimen.

In particular, the Lancet Oncology Commission recently presented newevidence on the issues involved in expanding global access toradiotherapy (Atun et al., Lancet Oncology 2015, 16, 1153-1186). Ithighlighted radiotherapy as a fundamental component of effective cancertreatment and control and that it is generally used sub-optimally. TheLancet editors pointed out that radiotherapy is more scalable than othertreatment modalities and is uniquely placed to deliver effectivecurative and palliative care (Coburn & Collingridge, Lancet Oncology,2015, 16, 1143). However, there are two continuing roadblocks totailoring radiotherapy to the needs of the individual patient.

First, and not until the present invention, there has been no“quantitative test” that measures the extent to which a given tumour, ina given patient, will respond to a given regimen of radiotherapy.

Second, there has been no “quantitative test” that measures the extentto which any tumour is likely to reoccur following radiation therapy.

Until the present invention, clinical decisions were based on medicalart, not quantitative measures to determine radiation responsiveness.

Without being bound to any particular theory, the inventor advances thefollowing explanation for the unexpected finding that ATI could be usedto predict radiation responsiveness:

ionizing radiation, such as gamma-rays and X-rays lead to the radiolysisof water which leads to the formation of the same type of chemicalentities in all cells, be those cells bacterial, algal, fungal,invertebrate, or the hundreds of cell types in a human body, or abnormalcell types which arise as a result of perturbations leading to cancerouscells.

Radiolysis universally generates the same types of reactive molecules,the three key ones being;

the hydroxyl radical OH., the superoxide radical O₂.⁻ and hydrogenperoxide (H₂O₂), (Daly, M; Nature Reviews Microbiology, 7, 237-245.2009).

In normal healthy mammalian cells that have not been irradiated, thesame three molecules are also formed as part of the normal mitochondrialrespiratory processes occurring via the mitochondrial electron transportchain. If oxygen receives less than its full complement of electrons,the result is the formation of O₂.⁻ and H₂O₂. If the H₂O₂ is not dealtwith immediately, any stray iron Fe²⁺ atoms will react with it andgenerate the highly dangerous hydroxyl radical OH.

In mammalian cells, the superoxide radical O₂.⁻ is handled bymitochondrial, cytosolic and extracellular enzyme systems, namelymanganese superoxide dismutase MnSOD located in the mitochondrion,copper-zinc superoxide CuZnSOD in the cytosol and extracellularsuperoxide dismutase ecSOD predominantly anchored to endothelial cells.

The H₂O₂ is cleared by both catalases and glutathione peroxidases toproduce water and molecular oxygen.

The highly dangerous hydroxyl radical OH. is not dealt with byenzymological processes. One example of the consequences of the highlevel of oxidative metabolism is in the mammalian brain, where it makesbrain cells very vulnerable to lipid peroxidation from OH.

The inventor reasoned that the chemical elements and levels of elementscontributes to radio-responsiveness, for example manganese. The inventornoted that whilst a whole body exposure of 10 Gray (Gy) is lethal tomost vertebrates, some bacteria such as D. radiodurans survive doses inexcess of 17,000 Gy. One contributing mechanism by which it may achievethis is that it accumulates 150 times more manganese and 3 times lessiron (Fe) than radiation sensitive bacterial species (Daly, M; NatureReviews Microbiology, 7, 237-245, 2009). Bacterial species with thehighest manganese-to-iron ratios are the most radioresistant, whereasthose with the lowest Mn/Fe ratios are hypersensitive. The mechanisticunderpinnings of radio-responsiveness reveal that manganese accumulationshields proteins with iron-sulphur (Fe-S) complexes from superoxideradicals such as (O₂.⁻) formed during irradiation. This shielding bymanganese prevents the release of ferrous ions (Fe²⁺) from iron-sulphurcontaining proteins, thus preventing the highly damaging interactions ofFe²⁺ with hydrogen peroxide. If Fe²⁺ manages to react with H₂O₂, theresult is an hydroxyl radical OH. which is dangerous and will oxidizealmost every type of biological molecule.

In contrast to hydrogen peroxide, O₂.⁻ does not easily cross membranesand hence builds up in cellular compartments. Thus any cellular systemthat can effectively shield Fe-S containing proteins from exposure tothe O₂.⁻ as well as minimizing the amount of Fe²⁺ available for theFenton reaction will minimize damage following irradiation and enhanceradio-resistance. The inventor reasoned that the bacterial data indicatethat the manganese ion is a protective metal and even at highconcentrations is largely innocuous to a bacterial cell, and likely welltolerated by many cell types of multi-cellular organisms. Conversely,any bacterial system that is low in manganese and high in free iron islikely less able to protect proteins and lipids from damage and theFenton reaction will ensure that the damaging OH. will increase itslevel leading to protein and lipid damage and death of the cell, hencecellular radio-sensitivity. A similar situation will pertain toeukaryotes.

The intracellular availability of free iron is known to play a key rolein irreversible protein damage via protein carbonylation. In yeast,carbonylation levels are increased when yeast lack a particular ironstorage protein, the homolog of which is the human mitochondriallylocated frataxin protein. Introduction of the human ferritin into such adefective yeast strain, partially restores the iron storage capacity ofsuch yeast, decreases free iron levels and counteracts the elevatedcarbonylation levels. Thus, iron storage proteins are likely to beimportant players in preventing cellular damage following ionizingradiation.

It is noted that although the superoxide radical O₂.⁻ is highly charged,it does not react with DNA. Rather, it reacts with selected targets,these being any exposed iron-sulphur (Fe-S) groups of certain proteins.

In summary, OH. is extremely damaging to all cellular components, butthe collateral damage it causes is restricted to a few Angstroms fromits site of formation owing to its short lifetime. H₂O₂ by contrast, candiffuse throughout the cell and reacts with Fe²⁺, yielding one of themost powerful oxidizing reactions known. This reaction produces more OH.The bacterial data reveal that responsiveness to ionizing radiation is acontinuous biological variable, which has multiple inputs: from cellularmanganese concentration and from the associated enzymology thatscavenges radicals, sequesters free iron, reduces hydrogen peroxide andminimizes and remanufactures proteins.

In vitro systems have demonstrated that the human manganese superoxidedismutase converts the superoxide radical O₂.⁻ to H₂O₂ and O₂. In anumber of human in vitro cellular systems, human MnSOD protein levelsand activity have been correlated with an increased resistance toionizing radiation. Similarly, lowering the level of MnSOD protein andactivity in cellular systems results in decreased radio-resistance.

Thus, in carefully controlled experimental circumstances, where thegenetic background of the experimental and control cells is keptconstant, and the only variable is either the introduced gene, itsantisense product, or an empty vector, the mitochondrially-located MnSODprotein helps to protect cells from the effects of ionizing radiation.

There are only three superoxide dismutases in human cells that handlethe superoxide radical O₂.⁻ and these superoxide dismutases all havedifferent cellular locations. Manganese superoxide dismutase MnSOD islocated in the mitochondrion, copper-zinc superoxide CuZnSOD is found inthe cytosol and the nucleus, while extracellular superoxide dismutaseecSOD is predominantly anchored to endothelial cells. The threesuperoxide dismutases deal with O₂.⁻ in these three different locations.

Whilst much attention has been focussed on superoxide dismutases, thebasic chemistry and biochemistry of chemical elements, such as Mn andCu, their location and usage within a cell is far more widespread thanthe specificity of superoxide dismutases e.g., Mn exists in complexformation (Daly et al., PLoS ONE, 5, e12570, 2010; Slade and Radman,Microbiology and Molecular Biology Reviews, 75, 133-191, 2011).

FIG. 2 illustrates three different manganese-binding entities. Manganeseforms complexes with orthophosphate and scavenges O₂.⁻. Manganese alsoforms complexes with free amino acids or peptides to scavenge anddecompose hydrogen peroxide, hydroxyl radicals (OH.) and O₂.⁻.Nucleosides, free amino acids and sundry organic metabolites scavengehydroxyl radicals (Slade and Radman, Microbiology and Molecular BiologyReviews, 75, 133-191, 2011).

Inorganic metals, such as, iron, copper, zinc and manganese are mostcommonly thought of as catalytic cofactors for proteins.

Notwithstanding the foregoing suggestion that metals play a role inradio-resistance, until the present invention, manganese has not beenquantified in a 2D cancerous context of a tumour compared to normal,whereby the radio-sensitivity/radio-resistance is determined andsubsequently utilized to firstly make decisions regarding radiationtreatment, and secondly, to predict the probability of tumourreoccurrence after radiation treatment.

All the hardware and software for carrying out the invention arecurrently available both commercially and clinically. It will beapparent to a person skilled in the art that technological variationsmay be useful. Preparation of the tissue sections, cells or populationsof cells for analysis according to the invention may be performedaccording to any known method in the art. For example, unstained tissuesections, unstained frozen sections, or cells deposited as a monolayervia a SurePath system are prepared according to the art and for example,as described herein. Any system available in the art may be used, e.g.,the SurePath system yields a monolayer of cells on a slide in a compactcircular area, and material prepared in this manner would be suitablefor LA-ICP-MS methodology. Any method known in the art may be used toprepare the test sample and/or control sample of the invention providedthe 2D integrity has not been lost. A number of ablation tracks can bemade, e.g. via laser ablation-Inductively coupled plasma-massspectrometry (LA-ICP-MS), laser ablation-time-of-flight-massspectrometry (LA-TOF-MS), inductively coupled plasma-optical emissionspectroscopy (ICP-OES), microwave plasma-atomic emission spectroscopy(MP-AES), laser induced break down spectroscopy (LIBS), secondary ionmass spectrometry (SIMS), X-ray absorption near edge structure (XANES),atomic absorption spectroscopy (AA) or X-ray fluorescence (XRF).

If the values within a single or multiple ablation tracks all falleither above, or below, a designated threshold, there is no need for anexamination by a pathologist. Conversely, for any tracks that areflagged by the computer software, then the companion stained slide isexamined by a pathologist.

Alternately, a slide is first examined by a pathologist, who woulddirect where the preferred ablation tracks should be done.

It should be noted that a single ablation track (of width less than 110microns) would ablate a 1 cm track of tumour tissue in about 70 seconds.In this respect, multiple tracks across a tumour sample, or multipledispersed areas within the tumour, located and designated by apathologist for subsequent ablation analysis, may be run in minutes withcurrent technology.

Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS),was originally introduced by Gray, Analyst, 110, 551-556. 1985,employing a ruby laser. This was subsequently superseded by solid stateNd:YAG and excimer-based lasers. The use of the former is described inHare, D, et al., Analyst 134, 450-453. 2009 (Table 1 of Hare et al. foroperational parameters). In regard to excimer lasers, the beam isgenerated by a gas mixture which is a combination of the noble gases(argon, krypton or xenon) with a reactive gas, such as chlorine orfluorine. Under high pressure and electrical stimulation, a pseudomolecule, (XeCl, KrF or ArF), termed exciplex, is created which givesrise to laser light in the ultraviolet.

A popular excimer-based system in the context of the LA-ICP-MS, is theAgilent 7700 ICP-MS coupled to a New Wave excimer generating a 193nanometer wavelength, the laser being first used by Gunther et al., J.Anal. At. Spectrom. 12, 939-944, 1977. The elemental analysis of freeions is performed on a LA-ICP-MS, e.g., using ICP-MS instrument AgilentTechnologies 7700 series which is interfaced with a New Wave ResearchExcimer 193 laser ablation unit. In addition, the ICP-MS Instrument isfitted with an octopole collision/reaction cell. Other ICP-MSinstruments are also available including Thermofisher's iCAP™ Q ICP-MS,Perkin Elmer's Nexion Series, Shimadzu ICP-MS 2030, and Tofwerk'sICP-TOF-MS. Instead of an ICP-MS system, the ATI may also be determinedwith atomic emission spectroscopy techniques such as inductively coupledplasma—optical emission spectroscopy (ICP-OES), microwave plasma—atomicemission spectroscopy, or laser induced break down spectroscopy (LIBS),or secondary ion mass spectrometry (SIMS), or X-ray absorption near edgestructure (XANES), or atomic absorption spectroscopy (AA), or X-rayfluorescence (XRF)

High purity liquid argon (Ar) is used as the carrier gas and plasmasource, while ultra-high purity (99.999%) hydrogen (H2) is used as thereaction gas. The LA-ICP-MS system is tuned on a daily basis and forboth standard mode and reaction mode using NIST 612 Trace Elements inGlass for maximum sensitivity and to ensure low oxide formation. Lowoxide production is assured by measuring a mass-to-charge ratio (m/z) of248/232 (representing ²³²Th¹⁶O⁺/²³²Th⁺) and is consistently less than0.3%. The instrument is fine-tuned for tissue analysis usingmatrix-matched tissue standards.

Typical operational parameters for the LA-ICP-MS system are given below,and for clarity, they are shown separately for the 7700 ICP-MS and forthe laser.

Icp-Ms Parameters.

The radio frequency power is 1250.0 Watts; the cooling gas flow rate is15.0 litres/minute; the carrier gas flow rate is 1.2 litres per minute,the sample depth is 4 millimetres; the quadrupole bias is −3.0 Volts;the octopole bias is −6.0 Volts; the dwell time is 62 milliseconds perm/z; extraction lens 1 is 5.0 Volts; extraction lens 2 is −100.0 Voltsand the hydrogen collision gas is 3.1 millilitres per minute.

Parameters of the New Wave 193 Excimer Laser.

Wavelength 193 nanometres; repetition frequency 40 Hertz; laser energydensity 0.3 to 0.5 Joules per square centimetre; spot diameter 35micrometres; line spacing 35 micrometres; monitored mass/charge ratio(m/z), 55 (Mn), 56 (Fe), 63 (Cu) and 66 (Zn). The raster speed is 140micrometres per second (four times 35 micrometres).

In brief, a glass microscope slide with an unstained section from aparticular tissue is placed in an ablation chamber and a high energylaser beam (of varying diameter) is focussed onto the section and someof the biological material is vaporized as a result of energy transferfrom the laser beam. The resulting particulate matter is moved by acarrier gas, which is usually Argon, Helium, or Argon plus Helium (inthe system used for one embodiment of this invention, it is Argon), intothe Inductively Coupled Plasma which, at a temperature exceeding 7,000degrees Celsius, but below 10,000 degrees Celsius, atomizes and ionizesthe particulate matter to its constituent elements. The use ofcollision/reaction cells (Tanner et al., Spectrochim. Acta. Part B, 57,1361-1452, 2002) minimizes spectral interferences, and “removes”polyatomic ions such as oxygen:argon species (¹⁶O⁴⁰Ar⁺) that wouldotherwise appear as a 56 signal and be incorrectly attributed to ⁵⁶Fe.In the dynamic reaction cell, interfering polyatomic ions are convertedto a different species at a higher m/z and no longer interfere with thetarget ion. Following emergence from the collision cell, the ions arefocussed into a quadrupole mass filter where they are separated by theirm/z ratios, and detected and quantified.

Instead of a quadrupole system, ICP-MS systems can incorporate a doublefocussing sector field mass spectrometer, or a Time-of-Flight (TOF)analyser. An ICP-TOF-MS has a very high throughput acquisition capacityof 30,000 full mass spectra per second (Resano et al., Mass Spectrom.Rev. 29, -55-78, 2010).

To generate a 2D elemental map of the material on a microscope slide,the laser is rastered across a sample from side to side, one track at atime from top to bottom, where the track has a chosen width. Theresulting image is visualized as adjacent pixels, but since the ablatedmaterial has depth, each pixel represents a volume of tissue, a voxel.

It will be apparent to the skilled artisan that the present inventionprovides the following advantages:

a) minimal sample preparation to avoid introduced artefacts;b) the ability to simultaneously and rapidly extract multi-elementaldata;c) the deconvolution of 2D information about the structure of the tumourin terms of its tumour cells, stromal cells, abnormal vasculature,transiting immune cells, and non-cellular material such as collagenbundles, all of which form the interacting milieu that constitutes thetumour and which impinges on its position between the lower boundary ofradiation sensitivity and the upper boundary of radiation resistance;d) it directly translates to the use of, or avoidance of, a therapeuticmodality, namely ionising radiation for any tissue or organ type. Whereradiation therapy is used, the invention determines the probability oftumour reoccurrence.e) it is not specific for a particular type of tumour. It is applicableto any tumour, localized or metastatic, benign or malignant. As such, itis pan-diagnostic. For example, a Prostate-Specific Antigen testmeasures a single circulating entity in the bloodstream which may beindicative of the presence of a benign or malignant tumour, or simplybenign prostatic hyperplasia, or strenuous exercise. As such it does notsuggest the type of therapeutic intervention that is required. It isalso specific to males. In contrast the present invention is notrestricted by specificity of gender or tumour type and hence there is noneed to develop specific antibodies or drugs to a particular tumourtype, such as Tarceva for non-small cell lung cancer.f) radiation, which can be external beam or implanted “seeds”, isdelivered to a localized anatomical region, unlike small molecularweight drugs, chemotherapeutics or antibody based biologicals which aredelivered via the vascular system, which spread to all tissues, andinvariably have off-target effects in normal tissues and cause variouslevels of toxicity, such as vemurafenib in melanoma, which induceskeratoacanthomas.g) the present invention is direct. It has no intermediate steps asregards multiple preparation steps for a sample. The assay is notconfounded by potential biases inherent in methods that rely for signalamplification on processes such as PCR, where the enzymes commonly usedin such procedures can introduce systematic bias through differentialrates of amplification of different sequences. There is no hybridizationof antibodies to tissue sections with its varying specificities ofhybridization and measurement of amplification signals. The presentinvention measures what is in a sample without the distortions thatoccur as a result of multi-step processing.h) there is a seven order linear dynamic range over which measurementscan be made. Both range and the linearity are crucial since they allow atrue measurement of elemental abundance in a cell population withoutintroducing potential errors by the prior art methods that requireconversion or amplification of entities.

The present invention is particularly suitable for detection of diseasestates, differentiation states of stem cells and derivative cellpopulations, detection or measurement of effects of medication on cellstate, and any other situation where an accurate indication of cellularstate is useful.

The present invention will now be described in more detail withreference to specific but non-limiting examples describing specificcompositions and methods of use. It is to be understood, however, thatthe detailed description of specific procedures, compositions andmethods is included solely for the purpose of exemplifying the presentinvention. It should not be understood in any way as a restriction onthe broad description of the inventive concept as set out above.

Example 1 Preparation of Tissue Standards

Normalisation and calibration experiments were run using 30 μm thicksections of matrix-matched tissue standards. These standards wereprepared from chicken breast tissue removed of any fat or connectivematerial, and were partially homogenized using an OmniTech TH tissuehomogenizer fitted with a polycarbonate probe (Kelly Scientific, NorthSydney, New South Wales, Australia), and subsequently spiked withstandard Ca, Mn, Fe, Co, Cu, and Zn solutions. Solutions were preparedusing high purity (min 99.995%) soluble chloride, sulfate, or nitratemetal salts (Sigma-Aldrich, Castle Hill, New South Wales, Australia)dissolved in 1% HNO₃ (Choice Analytical, Thornleigh, New South Wales,Australia) and diluted to concentrations of ca. 100,000 μg mL-1 and10,000 μg mL-1. Aliquots of the chicken breast were then spiked withvarying concentrations of each of the elements and homogenized at lowspeed for 5 min. Six ca. 250 mg aliquots of each homogenized tissuestandard were digested in 5:1 Seastar Baseline grade HNO₃/H₂O₂ (ChoiceAnalytical) in a Milestone MLS 1200 closed vessel microwave digester(Kelly Scientific) and analysed using solution ICP-MS to confirm theconcentration and homogeneity of each element in the tissue standards.The spiked tissue was frozen and sectioned into 30 μm sections andplaced onto glass microscope slides for analysis. This methodology canbe simply applied to sections cut at 5 microns, a small part of which isadded to each tumour slide.

Calibration and Background Analysis

The prepared matrix-matched tissue standards were used to constructcalibration curves for sensitivity normalisation of analytes underconditions described later for running the LA-ICP-MS. Data for a 10 speriod prior to ablation of the tissue were collected to obtain abackground signal for each m/z from the gas blank. This methodology wasused to allow for a comparison between different runs of differenttumours. For example, a 2K calibrated counts/second was achieved.

Sensitivity Threshold(s)

An alternate way of setting thresholds to that described earlier asstandard deviations relative to means and medians of a radiationsensitive control, or as empirically determined values from any tumourtype, is to set a sensitivity threshold by apportioning, for example,the testicular samples in the top histogram of FIG. 11, such that thethreshold is defined as a percentage of that histogram. For example, theseminomas of FIG. 11 derive from 55 patients and 41/55 of these patientsconstitute 75% of the histogram who fall below this 75% threshold, andall these patients are radiation sensitive. Next, 47/55 patientsconstitute 85% of the histogram and will be radiation sensitive.Finally, 52/55 patients constitute nearly 95 of the histogram, but someof these outlier patients are likely to have some degree of radiationresistance. These percentage figures representing a proportion of thehistogram are another way of expressing the variable threshold that canbe chosen for radio-responsiveness.

Example 2

In one example, a section of normal brain tissue on a microscope slideas described herein, was laser ablated and its metallomic contents wereanalyzed by the LA-ICP-MS system.

FIG. 3A shows a representative H&E stained 5 micron tissue section froma block of a normal human cortex from a 50 year old human female showingthe characteristics of normal cortical tissue. This Figure illustratesthe position of a three-track ablation (taken from the largermulti-track tissue ablation) that was carried out on an adjacentunstained section from the same block, but on a different slide. The H&Estained area shown in FIG. 3A corresponds to the equivalent unstainedarea analysed by LA-ICP-MS on the different slide. The unstained 5micron tissue section was cut from the same block, laser ablated andanalysed using the Agilent technologies 7700 LA-ICP-MS system, withdetailed operational parameters described previously. Unstained tissuesections are generally used in order to avoid the introduction of anychemical elements that occur in the stains, staining solutions or whichare leached from the containers during processing. Stained sections canalso be used.

To generate an elemental map of the unstained material on the separatemicroscope slide, the laser was rastered across the slide laterally, oneablation track at a time from top to bottom. Generally, this produces upto hundreds of horizontal tracks depending on the size of the tissuesample, with each track being 35 microns in width. Lateral resolution isa function of the integration time of the quadrupole mass spectrometerand was chosen in these data sets to give a resolution of 35 microns. Acomplete image was generated, which consists of a basic unit of a 35micron by 35 micron pixel. Since the tissue section is 5 microns thick,each pixel represents a 35×35×5 volume of tissue, namely a voxel of 6125cubic microns. An ablation run of 50 horizontal tracks, in which eachablation track consists of, say, 70 voxels, generated a 2D imageconsisting of 3500 voxels.

The calibrated signal of each of three metals ⁵⁵Mn, ⁶⁶Zn, and ⁵⁶Fe wasdetermined. The results are shown for each voxel of three contiguousablation tracks from a tissue section that ablated 70 voxels per track(FIG. 3B). The background-subtracted median values per voxel for thesemetals, expressed as calibrated counts per second, are 3,409, 6,651 and441,317 respectively. Measurements of ⁶³Cu were unavailable fortechnical reasons.

These raw data are presented in graphical form in FIG. 3C, where eachtrack consisted of 70 voxels. FIG. 3C shows graphs of raw numericalvalues of calibrated counts per second CC/S for the three metals, ⁵⁵Mn,⁶⁶Zn and ⁵⁶Fe in adjacent voxels in each of three ablation rows. Theunstained 5 micron tissue section was cut from the same block, laserablated and analysed using the Agilent technologies 7700 LA-ICP-MSsystem, with detailed operational parameters described below.

As the laser rasters across the unstained tissue section on the slide,the values for each of the metals change from background levels to thosecharacteristic of the tissue sample and then return to background valuesagain. The variation in values generally follows the morphologicalchanges and variations in tumour architectures seen in the stainedtissue sections.

In this Example, the raw free ion manganese values in the top panel,reveal that there is a spike in voxel 55 in ablation row two, while noneof the eight voxels contiguous to it show this large deviation from themedian (as seen from the data of FIG. 3B). These large single spikes foran individual voxel are due to machine “jitter” and such spikes arealmost always more than 3 standard deviations from the median value.While shown here for the illustrative purposes of raw data presentation,they will be removed prior to statistical analyses as they representmachine “noise”. They are sufficiently infrequent that even if theyremained part of the data analysis, they have a miniscule effect onimportant outcomes such as median values.

Example 3

Neoplastic Samples with Contrasting Radiation Resistances andSensitivities

As discussed above, the Lancet Oncology Commission highlightedradiotherapy as a fundamental component of effective cancer treatmentand control and that it is generally used sub-optimally (Atun et al.,Lancet Oncology 2015, 16, 1153-1186). Until the present invention, theclinically accepted norm is that some tumour types such as glioblastoma,mesothelioma and melanoma are largely radiation resistant, while mostlymphomas, small cell tumours of the lung, and tumours of the testis,such as classic seminomas, are radiation sensitive. Other major tumourtypes such as cancers of the breast and prostate are generally thoughtto be of “intermediate” radio-responsiveness, but “intermediate” iscontext dependent and difficult to determine. The clinical realityrelates to what percentage of primary cancers can be eradicated withradiotherapy alone and the dose that is required, as well as thepercentage susceptibility of various metastatic lessons to ablativeradiotherapy. Thus large seminoma and lymphoma masses are easilyeradicated with radiotherapy while sarcomas and pancreatic carcinomasare rarely eradicated, despite the dose. In addition, radiotherapy isconsidered “curative” for some “low/intermediate” risk prostate cancersand some prostate cancer metastases to the bone, and so such prostatecancers could certainly be classified as very radiosensitive.

Given the above clinical variation, we have chosen six tumour types forevaluating the ATI, as the first three provide the bookends forradiation resistance, and the second three provide the bookends forradiation sensitivity. It is clinically acknowledged, however, that evenwithin each of these six tumour types, there are a minority which do notrespond to radiation treatment in the expected manner. This is due toproblems with methods used prior to the present invention e.g., lowresolving power of H&E stained pathological material and theconsiderable disagreement among pathologists of the taxonomy within anytumour type. Even with the addition of antibody staining offormalin-fixed paraffin-embedded and Hematoxylin and Eosin (H&E) stainedtissue sections, the interpretation of the variation within a tumourtype remains subjective and the concordance among pathologists isvariable (Elmore et al., JAMA, 313; 1122-1132. 2015).

Melanoma illustrates the challenges of personalizing radiotherapies.Historically it has been considered to be intrinsically radio-resistant,a perception originating from early cell culture studies and analyses ofsurvival curves. This belief has recently been evaluated on the basis ofdata from 4 decades of the clinical use of radiation therapy in melanomapatients (Mahadevan et al., Oncology, 29, (10): 743-751, 2015). Thenewer interpretation is that the radio-responsiveness of melanoma isdiverse. It is accurately summarized by Burmeister; “I've been workingwith melanoma for over 25 years and it still amazes me how in somepatients the disease just melts away and in others it just laughs at youand kills the patient within a few weeks or months . . . there is anincredible variation in the behaviour of this disease in individualpatients.”

As the atomic data presented herein reveal, there is now, for the firsttime, (i) a quantitative underpinning of which melanoma patients, (aswell as other tumour types) are suitable candidates for radiotherapy,(ii) a quantitative basis for determining in which patients a cancer islikely to re-occur after radiotherapy, and (iii) a measurable basis fortumour variation. It should also be noted that such heterogeneity is notconfined to melanoma. Breast and prostate cancer patients and theirtumours are also heterogeneous at multiple levels, one being theirresponse to radiation treatment.

1. Radiation Resistant Tumours

In another Example, the metallomic values of neoplasms that areconsidered to be at the radiation resistant end of the clinical spectrumwere analyzed. For example, a deadly brain neoplasm, glioblastomamultiforme, which is considered to be at the radiation resistant end ofthe clinical spectrum, was analysed as described in Example 2. Thisexample illustrates how the metallomic values of a brain neoplasm differfrom those of normal brain tissue, when both the normal and neoplasticsamples are present on the same slide and are evaluated on the sameLA-ICP-MS system under the same experimental run conditions.

FIG. 4A shows a representative H&E stained 5 micron tissue section froma block of a 19 year old human female with a brain neoplasm classifiedas glioblastoma multiforme. The H&E stained area shown in the Figurecorresponds to the equivalent unstained area analysed by LA-ICP-MS on adifferent slide.

The calibrated signal of each of three metals ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe wasdetermined and is shown for each voxel of three contiguous ablationtracks from a tissue section that ablated 68 voxels per track (FIG. 4B).The background-subtracted median values per voxel for these metals,expressed as calibrated counts per second, were found to be 3,198, 5,066and 219,905 respectively. A graph of raw numerical values of calibratedcounts per second for the three metals, ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe in adjacentvoxels in each of three ablation rows from the human female withglioblastoma multiforme was prepared and is shown in FIG. 4C.

To obtain a measure of the variation between different sections takenfrom the same block of the same human subject with glioblastomamultiforme, two unstained areas on the same slide were analysed byLA-ICP-MS in the same machine run. The results are shown in FIGS. 5A, 5Band 5C.

FIG. 5A shows a representative H&E stained 5 micron tissue section froma different region of the same block of the 19 year old human femalewith glioblastoma multiforme shown in FIG. 4.

The calibrated signal of each of three metals ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe wasdetermined for each voxel of three contiguous ablation tracks from atissue section that ablated 69 voxels per track (FIG. 5B). Thebackground-subtracted median values per voxel for these metals,expressed as calibrated counts per second, were found to be 2,980,4,155, and 190,127 respectively. A graph of raw numerical values ofcalibrated counts per second for the three metals, ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fein adjacent voxels in each of three ablation rows from the human femalewith glioblastoma multiforme was prepared and is shown in FIG. 5C.

These results demonstrate that variation in background-subtracted mediancalibrated counts per second in the same LA-ICP-MS machine run, fordifferent tissue sections taken from the same block of the sameindividual is excellent. For ⁵⁵Mn the CC/S values were 3,198 and 2,980;for ⁶⁶Zn the CC/S values were 5,066 and 4,155, and for ⁵⁶Fe the CC/Svalues were 219,905 and 190,127 respectively.

The next example is of a different neoplasm, which is also considered tobe at the radiation resistant end of the clinical spectrum. This exampleis a neoplasm of mesothelial origin and comes from a 60 year old malewith malignant mesothelioma of the abdominal cavity, and itscharacteristics are illustrated in FIGS. 6A, 6B and 6C.

FIG. 6A shows a representative H&E stained 5 micron tissue section froma 60 year old human male with malignant mesothelioma.

The calibrated signal of each of four metals ⁵⁵Mn, ⁶⁶Zn, ⁵⁶Fe and ⁶³Cuwas determined for each voxel of three contiguous ablation tracks from atissue section that ablated 67 voxels per track (FIG. 6B). Thebackground-subtracted median values per voxel for these metals,expressed as calibrated counts per second are 4,522, 8,805, 112,772 and1,097 respectively. A graphical representation of the raw numericalvalues of calibrated counts per second for the four metals, ⁵⁵Mn, ⁶⁶Zn,⁵⁶Fe and ⁶³Cu in adjacent voxels in each of three ablation rows from the60 year old human male with malignant mesothelioma was determined and isshown in FIG. 6C.

A further example of a different neoplasm, which is also considered tobe at the radiation resistant end of the clinical spectrum, comes from a50 year old male with malignant melanoma of the esophagus (stage IIa,T3N0M0). The metallomic characteristics are illustrated in FIGS. 7A, 7Band 7C.

FIG. 7A shows a representative H&E stained 5 micron tissue section froma 50 year old male with malignant melanoma of the esophagus.

The calibrated signal of each of three metals ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe wasdetermined for each voxel of three contiguous ablation tracks from atissue section that ablated 68 voxels per track (FIG. 7B). Thebackground-subtracted median values per voxel for these metals,expressed as calibrated counts per second are 10,565, 6,961, and 121,495respectively. A graphical representation of the raw numerical values ofcalibrated counts per second for the three metals, ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fein adjacent voxels in each of three ablation rows from the 50 year oldhuman male with malignant melanoma of the esophagus was determined asshown in FIG. 7C.

It should be noted that there is much more variation in metallomiccontent between voxels in some melanomas than in normal tissues and inother neoplasms. One of the major contributors to this increase invariation is the presence of intracellular and extracellularinhomogeneities in the distribution of melanin, an entity that hasstorage capacity for different chemical elements, particularly metals.This morphological inhomogeneity is clearly visible in the first figurepresented in this application, which is part of an H&E stained sectionfrom a stage Ib malignant melanoma of the neck of a 48 year old male(FIG. 1). Within the same tumour, there was an amelanotic lineage and ahighly melanised different area. The metallomic characteristics of thistumour are further described herein (see Example 5).

2. Radiation Sensitive Tumours

The characteristics of three different types of neoplasms that areconsidered to be at the radiation-sensitive end of the clinical spectrumwill now be described. They are diffuse B-cell lymphomas, small cellcancers of the lung, and seminomas of the testis.

Lymphomas: the first example of a neoplasm considered to be at theradiation sensitive end of the clinical spectrum, comes from a 57 yearold male with diffuse B-cell lymphoma in the testis. Its metallomiccharacteristics are illustrated in FIGS. 8A, 8B and 8C.

FIG. 8A shows a representative H&E stained 5 micron tissue section froma 57 year old male with diffuse B-cell lymphoma.

The calibrated signal of each of three metals ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe wasdetermined for each voxel of three contiguous ablation tracks from atissue section that ablated 79 voxels per track (FIG. 8B). Thebackground-subtracted median values per voxel for these metals,expressed as calibrated counts per second are 776, 18,892, and 247,923respectively. A graphical representation of the raw numerical values ofcalibrated counts per second for the three metals, ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe,in adjacent voxels in each of three ablation rows from the 57 year oldmale with diffuse B-cell lymphoma in the testis was prepared and isshown in FIG. 8C. The Y axis shows calibrated counts per second (CC/S)of that metal ion, while the X axis shows the voxel number of the threeablation tracks.

Malignant carcinoma: The second example of a neoplasm considered to beat the radiation sensitive end of the clinical spectrum, comes from a 38year old male with a small cell undifferentiated malignant carcinoma ofthe lung (stage IIIb, T4N1M0). Its metallomic characteristics areillustrated in FIGS. 9A, 9B and 9C.

FIG. 9A shows a representative H&E stained 5 micron tissue section froma 38 year old male with a small cell undifferentiated malignantcarcinoma of the lung.

The calibrated signal of each of three metals ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe wasdetermined for each voxel of three contiguous ablation tracks from atissue section that ablated 65 voxels per track (FIG. 9B). Thebackground-subtracted median values per voxel for these metals,expressed as calibrated counts per second are 1,749, 4,836, and 88,317respectively. A graphical representation of the raw numerical values ofcalibrated counts per second for the three metals, ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe,in adjacent voxels in each of three ablation rows from the 38 year oldmale with a small cell undifferentiated malignant carcinoma of the lungwas prepared and is shown in FIG. 9C.

Seminoma: the third example of a neoplasm considered to be at theradiation sensitive end of the clinical spectrum, comes from a 52 yearold male with seminoma. Its metallomic characteristics are illustratedin FIGS. 10A, 10B and 10C.

FIG. 10A shows a representative H&E stained 5 micron tissue section froma 52 year old male with seminoma.

The calibrated signal of each of three metals, ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe, wasdetermined for each voxel of three contiguous ablation tracks from atissue section that ablated 79 voxels per track (FIG. 10B). Thebackground-subtracted median values per voxel for these metals,expressed as calibrated counts per second are 1,565, 13,528, and 52,115respectively. A graphical representation of the raw numerical values ofcalibrated counts per second for the three metals, ⁵⁵Mn, ⁶⁶Zn and ⁵⁶Fe,in adjacent voxels in each of three ablation rows from the 52 year oldmale with seminoma was prepared and is shown in FIG. 10C.

In toto, the above examples demonstrate the metallomic characteristicsof three types of neoplasm that are at the radiation resistant end ofthe clinical spectrum, (glioblastoma, mesothelioma and melanoma), andthree types of neoplasms that are at the radiation sensitive end of theclinical spectrum (B-cell lymphomas, small cell cancers of the lung andseminomas of the testis).

Example 4 Application of the LA-ICP-MS Method to Patient Cohorts

While the above examples are illustrative of the metallomiccharacteristics of single individuals in each category of cancerpatients, the in-depth analysis of 55 individuals with seminoma, 10 withlymphoma, 20 with small cell lung cancer, 64 with melanoma, 25 withglioblastoma multiforme or astrocytoma, and 10 with mesothelioma,revealed a dichotomy in regards to their total manganese content and theknown clinical outcomes of these cancer types to radiotherapy, wasdetermined and is shown in FIG. 11. The total median manganese free ioncontents (expressed as calibrated counts per second, CC/S of ⁵⁵Mn), intumours from patients with cancers of the testis (seminoma), lymphoma,small cell carcinoma of the lung, mesothelioma, brain (glioblastomamultiforme and astrocytoma) and melanoma, were determined and are shown.Each square represents a single patient, except in the case of twomelanomas where two of the 64 patients each had two major lineageswithin their tumours, and each lineage was represented separately in thehistogram with a square.

The accepted clinical situation is that approximately 85% of seminomas,lymphomas and small cell carcinomas of the lung are sensitive toradiation, resulting in a great reduction, and sometimes completeelimination, of tumours and increasing life expectancy. Consistent withthese values and as determined herein, 89% of the individuals withseminoma, 80% of those with small cell carcinoma of the lung and 90% ofthose with lymphoma, were found to have total tumour manganese contentsthat fall below 2,000 CC/S. In this respect, this is the firstdemonstration of the correlation of such radio-sensitive tumours havingvalues that fall below a threshold of 2,000 CC/S.

In contrast to the above, the three tumour types that are considered tobe largely resistant to radiation (mesotheliomas, glioblastomamultiforme, astrocytomas and melanomas), and which have a proportionthat are differentially sensitive to it, present a different outcome.Not only is the variance within each of these three tumour types greatlyincreased, but 90% of the mesotheliomas, 85% of the glioblastomamultiforme, astrocytomas, and 59% of the melanomas, fall above the 2,000CC/S manganese value.

Given the above dichotomy, the metallomic content e.g., the manganesecontent of tumours becomes a critical indicator for radiation therapythat has simply not been previously recognized in terms of practicalclinical care until the present invention. In these examples using a 35micron pixel, individuals having tumours with a low total manganesecontent (e.g., below 2,000 CC/S) are highly likely to benefit fromradiation therapy, whereas those with a manganese content (e.g.,significantly above 2,000 CC/S) are likely to benefit by avoidingradiation therapy. As pixel size is increased, the CC/S will increase,and hence a higher threshold is contemplated. As the range of valuesremains well within linearity, there are no statistical issues that areproblematic.

Given that the majority of cancer patients receive radiation therapybased on medical art, rather than quantitative data relevant to tumourcharacteristics, such data support the use of quantitative data usinge.g., LA-ICP-MS to clinical practicality. Medical art relates to whatpercentage of primary cancers can be eradicated with radiotherapy aloneand the dose that is required to do so, as well as the percentagesusceptibility of various metastatic lesions to ablative radiotherapy.As a population-based approach it does not apply to the tumour(s) of aspecific patient and is not “personalized medicine”.

Statistical Analysis

The two sample nonparametric Kolmogorov-Smirnov (K-S) test compares thecumulative distributions of two data sets. The null hypothesis is thatboth data sets were sampled from populations with the same distribution.

From the data above, a comparison of the distribution of values fortesticular cancer (seminoma) versus lymphoma yielded a D value of0.2182, which has an associated non-significant P value of 0.762;seminoma and lymphoma were not significantly different and were sampledfrom populations with the same distribution.

A comparison of the distribution of values for testicular cancer(seminoma) versus small cell lung yielded a D value of 0.3182, which hasan associated non-significant P value of 0.081; seminoma and small celllung were not significantly different and were sampled from populationswith the same distribution.

A comparison of the distribution of values for small cell lung versuslymphoma yielded a D value of 0.3000, which has an associatednon-significant P value of 0.507; small cell lung and lymphoma were notsignificantly different and are sampled from populations with the samedistribution.

Conclusion: the data from seminoma, lymphoma and small cell lungrepresented a statistically valid unitary grouping.

A comparison of the distribution of values for brain cancers(glioblastomas and astrocytomas) versus mesotheliomas yielded a D valueof 0.3250, which has an associated non-significant P value of 0.371;brain cancers and mesotheliomas were not significantly different andwere sampled from populations with the same distribution.

A comparison of the distribution of values for brain cancers(glioblastomas and astrocytomas) versus melanomas yielded a D value of0.2226, which has an associated non-significant P value of 0.277; braincancers and melanomas were not significantly different and were sampledfrom populations with the same distribution.

A comparison of the distribution of values for melanomas versusmesotheliomas yielded a D value of 0.4303, which has an associatednon-significant P value of 0.056; melanomas and mesotheliomas were notsignificantly different and were sampled from populations with the samedistribution.

Conclusion: brain cancers, mesotheliomas and melanomas are astatistically valid unitary grouping.

Comparison Between Groups

A comparison of the distribution of values for testicular cancer(seminoma) versus brain cancers yielded a D value of 0.7712, which hasan associated significant P value of 0.000; seminoma and brain cancerwere significantly different and were sampled from populations withdifferent distributions.

A comparison of the distribution of values for testicular cancer(seminoma) versus mesothelioma yielded a D value of 0.8455, which has anassociated significant P value of 0.000; seminoma and mesothelioma weresignificantly different and were sampled from populations with differentdistributions.

A comparison of the distribution of values for testicular cancer(seminoma) versus melanoma yields a D value of 0.6879, which has anassociated significant P value of 0.000; seminoma and melanoma aresignificantly different and are sampled from populations with differentdistributions.

A comparison of the distribution of values for lymphoma versus braincancers yields a D value of 0.7750, which has an associated significantP value of 0.000; lymphoma and brain cancer were significantly differentand were sampled from populations with different distributions.

A comparison of the distribution of values for lymphoma versusmesothelioma yields a D value of 0.9000, which has an associatedsignificant P value of 0.000; lymphoma and mesothelioma weresignificantly different and were sampled from populations with differentdistributions.

A comparison of the distribution of values for lymphoma versus melanomayielded a D value of 0.6727, which has an associated significant P valueof 0.000; lymphoma and melanoma are significantly different and aresampled from populations with different distributions.

A comparison of the distribution of values for small cell lung versusbrain cancers yielded a D value of 0.7333 which has an associatedsignificant P value of 0.000; lymphoma and brain cancer weresignificantly different and were sampled from populations with differentdistributions.

A comparison of the distribution of values for small cell lung versusmesothelioma yielded a D value of 0.9000, which has an associatedsignificant P value of 0.000; lymphoma and mesothelioma weresignificantly different and were sampled from populations with differentdistributions.

A comparison of the distribution of values for small cell lung versusmelanoma yielded a D value of 0.6273, which has an associatedsignificant P value of 0.000; lymphoma and melanoma were significantlydifferent and were sampled from populations with differentdistributions.

Conclusion: seminomas, lymphomas and small cell lung cancer were foundto be a unitary grouping that is statistically distinct from the unitarygrouping of brain cancers, mesotheliomas and melanomas.

Whilst the statistical analysis indicates lymphoma, small cell lung,brain, and mesothelioma as being drawn from a normal distribution, suchis not the case for the melanomas and the seminomas, neither of whichconform to a normal distribution nor to a log normal distribution.

A statistical analysis using the Kolmogorov-Smirnov test indicates thatthe distribution of melanoma values depicted in FIG. 11 neither conformsto a normal distribution, nor a log normal distribution. As shown inlater examples, the melanomas exhibit a heterogeneity that is increasedby intra- and extra-cellular deposits of melanin, a heterogeneous set ofpolymers which bind multiple metals, and by the presence of HighMetallomic Regions (HMRs), distributed within many melanomas. These HMRsare a source of hidden atomic variation that has been uncovered for thefirst time owing to the technology described in this application. Suchregions are undetectable by conventional pathological examination.

It is likely that the seminomas in FIG. 11 consist of two populations,one with a small variance that makes up the bulk of the individuals andone that is more diverse and extends from 2K to 4K CC/S. It is probablethat the bulk of the seminomas in FIG. 11 correspond to the clinicalgroup that has an excellent response to radiation, whereas the remainingsubgroup is relatively more radioresistant. This finding is consistentwith clinical data (below). It should also be noted that the seminomasdepicted in FIG. 11 are a unitary group, namely “classic” seminomas in apathological context. Despite this outwardly appearing morphologicalhomogeneity, the metallomic analysis has revealed an underlyingheterogeneity that is hidden from conventional analyses.

1. Cancers of the Testis

For tumours of the testis, the major division is relativelystraightforward with testicular Germ Cell Tumours (GCT) falling into twolarge categories, seminoma and non-seminoma. For patients presentingwith testicular “cancer”, approximately 50% are diagnosed with seminoma.Of these, approximately 85% present with stage I disease with theremainder being clinical stage IIA and IIB. (The distinction betweenstage IIA and stage IIB, is that lymph nodes are 2 cms in the former and2 to 5 cms in the latter).

Postoperative radiation treatment for testicular seminoma has been themainstay of adjuvant therapy for more than half a century. Seminomas areone of the most sensitive tumour types to radiation, where a clinicaltrial of stage I testicular seminoma reveals that treatment doses of 20Gray, given as 10 fractions over 2 weeks, is sufficient to lead toalmost 98% cure rates at 5 years (Jones et al., Journal of clinicalOncology, 23, 1200-1208, 2005; Medical Research Council TrialTE18/European Organisation for the Research and Treatment of CancerTrial 30942 (ISRCTN18525328)). This radiation dose compares to 60 Gy(and up to 90 Gy), for high grade gliomas, such as glioblastomamultiforme, where overall survival is increased only by months.

The extreme radio-sensitivity of early stage seminoma is well described,with dose reductions being taken as low as 13 Gy for testicularintraepithelial neoplasia, which is a precursor to a more invasive formof cancer (Sedlmayer et al., Int J Radiat. Oncol. Biol. Phys. 50,909-913, 2001; Classen et al., Br J Can. 88, 828-831, 2003). TheEuropean Germ Cell Consensus Group summarized its position in 2008, with20 Gy in single doses of 2 Gy each being the recommended radiotherapy(Krege et al., Eur. Urol. 53, 478-496, 2008). The authors point out thatfor stage II testicular seminoma, a total dose of 30 Gy for stage IIAand a total dose of 36 Gy for stage IIB “seems reasonable”. Thestatement that these two dosages “seem reasonable” highlights theinexactness of the prior art (Krege et al., Eur. Urol. 53, 497-513,2008), It should also be noted that although 36 Gy is the recommendeddose, it has been pointed out earlier for stage II by Classen et al., JClin Oncol 21, 1101-1106, 2003), that there is a potential for dosereduction. It should also be noted that relapse-free survival for stageIIA is 95%, and for stage IIB it is 89%. Overall survival is close to100%.

The American recommendations for stage I and stage II seminomas are inline with the European ones described above. Up to 2015, Mead et al.,evaluated the evidence from a number of clinical trials Mead et al.,Journal of the National Cancer Institute, 103, 241-249, 2011) and therecommendations for stage I seminoma are as above. For stage IIAseminomas, low dose radiotherapy to the paraaortic lymph nodes andsuperior ipsilateral pelvis is recommended with a total dose of 25 to 35Gy in areas of gross adenopathy.

The exquisite sensitivity of germ line tumours is put into perspective,by comparing the radio-sensitivity of testicular cancers with those ofbrain cancers.

2. Cancers of the Brain

There is considerable variation in clinical practice for the managementand treatment of adult gliomas, which in Australia and New Zealand occurwith the following frequencies; 4% astrocytoma grade I; 10% astrocytomagrade II; 22% astrocytoma grade III; 52% glioblastoma multiforme gradeIV; and with oligodendrogliomas and oligoastrocytomas making up theremainder (Cancer Council Australia/Australian Cancer Network/ClinicalOncological Society of Australia, ISBN 978-0-9775060-8-8; 2009). Ofinterest for this invention is glioblastoma multiforme, the mostadvanced of these gliomas, as it is considered by radiologists to beradiation resistant. In addition, the comparison of glioblastoma withstage III astrocytomas is revealing in terms of their metallomics, asagainst the clinical comparisons (see below). The standardradiotherapeutic treatment for high-grade astrocytomas is 60 Gy in 2 Gyfractions (sometimes with a 10 Gy boost), but there are no data in whichthe optimal dose has been determined for grade IV gliomas such asglioblastoma multiforme. For grade III gliomas many radiologists use59.4 Gy with a fractionation procedure of 1.8 Gy, on the “expectation”that the 10% reduction per fraction from 2.0 Gy to 1.8 Gy in the case ofgrade III gliomas may cause less tissue damage.

Two phase III clinical trials bear on these data; the Radiation TherapyOncology Group (RTOG7401) and the Eastern Cooperative Oncology group(ECOG1374) (Nelson et al., NCI Monogr. 6, 279-284, 1988 and Chang etal., Cancer, 52, 997-1007, 1983). There were no survival differencesbetween treatment arms (radiation; radiation plus radiation boost;radiation plus BCNU, and radiation plus CCNU) and little differencebetween anaplastic astrocytoma and glioblastoma. It is salient in thecontext of minor differences in survival outcome between anaplasticastrocytoma and glioblastoma, that the metallomic data for thesecategories are heterogeneous, their distributions overlap, and they arenot significantly different from each other when tested using theKolmogorov-Smirnov test (P=0.581).

The central issue with radiation therapy to the whole brain is theoccurrence of multiple complications such as neurocognitive problems,leukoencephalopathies and endocrinopathies. While some of these havebeen ameliorated with Involved Field Radiation Therapy (IFRT), thefrequency of relapse has not altered, nor the percentage of patientswith multilocal failures.

There is clearly a need to better personalize therapies for patients,e.g., radiotherapy to those patients who are at the radiation sensitiveend of the spectrum, while sparing those who are likely to be radiationresistant. The current one-size-fits-all therapy, e.g., radiationtherapy, is obsolete, given the new knowledge provided by the date ofthis application including the metallomic data.

3. Cancers of the Serosal Membranes; Mesotheliomas

Mesotheliomas are considered to be radiation resistant, and treatmentprotocols are of the order of a dose of 54 Gy delivered in 1.8 Gyfractions.

The clinical situation with mesotheliomas is little different to thatconfronting physicians with other tumour types. In first examiningsurgically resectable tumours, there is the classic trimodality: adefinitive surgical procedure, radiation therapy and systemicchemotherapy. In the case of mesotheliomas, the role of definitivesurgery is controversial and it is unknown whether resection of a tumouryields an improvement in survival for that particular patient and noprospective clinical trials bear on this matter. There are also noadequately powered randomized phase III clinical trials that bear on theintegration of radiation therapy and chemotherapy before and or aftersurgery, the closest being a phase II trial using a hemithoracicradiation therapy of 54 Gy following extrapleural pneumonectomy, (Ruschet al., J Thorac Cardiovasc Surg 122, 788-795, 2001).

4. Small Cell Lung Cancer

Radiologists and physicians consider small cell lung cancer to be aradio-sensitive tumour with good response rates to radiotherapy. TheEuropean Society of Medical Oncology (ESMO) has documented its modifiedTumour Node Metastasis classification and stage grouping, and releasedits Guidelines for diagnosis, treatment and follow up (Fruh et al.,Annals of Oncology, 24, (Supplement 6), vi105, 2013; Table 3). The bestoverall survival outcome with curative intent is from a total dose of 45Gy, with daily 1.5 Gy fractions, together with chemotherapy (Turrisi etal., N Engl J Med, 340, 265-271, 1999).

5. Lymphomas

The Cancer Council Australia and the Australian Cancer Network, have setout clinical practice guidelines for the diagnosis and management oflymphoma, (ISBN: 0-9775060-0-2; 2005). For clinical stage I-III lowgrade follicular lymphoma it recommends involved-field radiation therapyof 30-36 Gy. For adult non-Hodgkin's lymphoma, the US National CancerInstitute recommends doses between 25 Gy and 50 Gy. For the diffuseB-cell lymphomas described herein, the National Comprehensive CancerNetwork (NCCN.org; 2015) recommends 30-36 Gy after chemotherapy, 45-55Gy as primary treatment for refractory to chemotherapy, ornon-candidates for chemotherapy, and 30-40 Gy for salvage pre- orpost-stem cell transplantation. In the varied spectrum that comprisesnon-Hodgkin's lymphoma, some patients have tumours that remain indolentfor long periods of time, others evolve rapidly and require immediatetreatment. As can be seen from the above in terms of radiation therapy,it is largely a one-size-fits-all situation.

6. Melanomas

Melanomas have generally been considered to be a radioresistant tumour,but the data are conflicting, with much of the evidence deriving fromcell lines that have demonstrated a wide spectrum ofradio-responsiveness. The clinical practice guidelines for themanagement of melanoma in Australia and New Zealand have been set out indetail by the Cancer Council Australia/Australian CancerNetwork/Ministry of Health New Zealand. For Australia see ISBN978-0-9775060-7-1 and for New Zealand see ISBN (electronic)978-1-877509-05-6. The clinical data have been reviewed by Wazer et al.,UptoDate, 2015 and they too are conflicting. The clinical trialsreviewed by Wazer et al., led them to conclude that melanoma is aradio-responsive tumour, “but the optimal dose and fraction remainuncertain”. In terms of metastatic disease, there are substantialdifferences in radio-responsiveness between cutaneous, lymph node,visceral metastases or metastases to bone or brain. (It should be notedthat the conflicting clinical trial data make better scientific sense interms of the metallomic data, where there is a wide range of manganesevalues between patients, and as revealed in FIG. 1, large differenceswithin a single tumour). Depending on the site of melanoma, doses havevaried from 32 Gy up to 100 Gy. Finally, while radiation therapy isuseful in palliative care in the settings of bone, brain and visceralmetastases, it is quite unclear whether large dose fractions improvepalliation.

Example 5 Melanin and Morphological Inhomogeneities and RadiationSensitivity/Insensitivity

As described above, the distribution of melanin, which has storagecapacity for different chemical elements, particularly metals, can be amajor contributor to variations seen with melanomas. FIG. 1 is an imageof an H&E stained melanoma which shows this morphologicalnon-homogeneity, which is clearly visible as two distinct lineageswithin it; one lineage is light coloured and largely amelanotic and theother is darkly staining and contains both intracellular andextracellular melanin. In addition, the cells of the amelanotic regionare more uniform in terms of their nuclear sizes and morphologies, thancells in the heavily melanized regions, a finding that impinges on theATI. The lower the variance of a population of cells in any of multiplecharacteristics, the more restricted will be its response to anyperturbogen. The metallomic content of the top part of the same tissuesection as depicted in FIG. 1 was measured. A two-dimensionalrepresentation of the ablation tracks taken through the melanoma isshown in FIG. 12. These data illustrate the variation in manganese levelin different parts of the same tumour and emphasize the importance ofcharacterizing a 2D landscape in order to make informed choices onwhether a patient should be radiation treated, or not. Thebackground-corrected median values for manganese in thesevisually-selected areas are 31,939 calibrated counts per second and3,045 calibrated counts per second.

The 2D landscape provides an estimate, within a tumour, of theproportion of a sampled area that exists above a designated threshold.In the above sample, this can be defined, for example, as to whatproportion of the landscape falls below the 10^(th) percentile, the20^(th) percentile, the 30^(th) percentile, the 40^(th) percentile the50^(th) percentile etc. and accordingly, quantitation is achieved. Thus,for example, if 90 percent of the area sampled is very low in manganese,then the tumour as a whole is likely to be susceptible to radiation. Onthe other hand, there is little point in irradiating a tumour in which90% of the area is high in manganese, as radiation will leave most ofthe tumour intact. Note that these percentiles cannot be derived from asample of a tumour that has been “ground up” for molecular work.

The same tumour of FIG. 1 was analyzed further and the content of all 4metals, ⁵⁵Mn, ⁶⁶Zn, ⁵⁶Fe and ⁶³Cu, was determined and shown in FIG. 13.The dichotomy, as seen morphologically, is visible from these results.As the ablation travels from the left hand side with very high values,to the right hand side with far lower values, the inflexion point occursaround voxel 34.

On the other hand, melanomas with uniform morphology, may still displayvery different metallomic content. For example, FIG. 14 shows an H&Estained tissue section of a melanoma from the left forefinger of a 54year old male: stage IV, T4N0M1. This particular melanoma has a uniformmorphology and the pathology would appear unremarkable to an experiencedpathologist (FIG. 14, upper panel). The metallomic content was measuredin this melanoma, and is shown in FIG. 14, lower panel. The underlying2D metallomic content is very different. Indeed, there are 2 largeconcentrations of cells with very high levels of manganese.

The median ⁵⁵Mn value for the bulk of this tumour sample was determinedto be 817 background corrected calibrated counts per second, whichplaces this tumor in the very sensitive end of the radio-responsivenessspectrum. On the other hand, the larger of the two whitish flat areas,namely the HMR, was found to have a value exceeding 6,500 calibratedcounts per second, placing it at the radio-resistant end of thespectrum.

These two regions are apparent from visual inspection of a voxel matrix.However a rigorous analysis of thresholds is necessary for an in-depthanalysis of these two HMR regions and this analysis is illustrated inFIGS. 15 and 16. As used herein, when referring to a specific metal in aHigh Metallomic Region (HMR), the HMR is designated as e.g., HMR(⁵⁵Mn),HMR(⁶⁶Zn), HMR(⁵⁶Fe) and HMR(⁶³Cu) respectively, with HMR(^(A)M)referring to the generic case of Any Metal.

Panel A of FIG. 15 shows the uniform morphological nature of a tumourarea of 22×60 voxels (sampled from an approximately 1800 voxel area) asvisualized by standard H&E pathology. Panel B shows the two HMR(⁵⁵Mn)regions as found by visual inspection of the numbers in each voxel.Panel C shows the HMRs that emerge from this 22×60 voxel 2D landscapewhen one uses as a threshold the median value per voxel of the wholearea plus the standard deviation (St.Dev.) of the voxels of the wholearea (median+St.Dev.). A different threshold method also robustlyuncovers the same two regions in this 22×60 voxel 2D landscape (PanelD), this second method utilizes two times the median of the voxel valuesof the whole area (2× median).

As seen by the distribution of clustered voxels that appear abovethreshold in panels C and D (black squares in a background of greysquares), the outcome of using (median plus St.Dev.) versus (2× median),is near identical in terms of finding HMRs(⁵⁵Mn). Analysis of the sixtumour types reveals that the 2× median method may be more consistentthan the median plus St.Dev. method in detecting HMRs(⁵⁵Mn), HMRs(⁶⁶Zn),HMRs(⁵⁶Fe) and HMRs(⁶³Cu). Whilst it will be apparent to the skilledartisan that either method may be used according to the invention, byway of non-limiting example, the 2× median methodology has been used insubsequent analyses shown herein.

A further important finding relating to the distribution of HMR(⁵⁵Mn)and HMR(⁶⁶Zn) in the same atomic landscape, is illustrated in FIG. 16.The top panel shows the identical H&E tumour landscape of FIG. 15, themiddle panel shows the two detected HMR(⁵⁵Mn) areas, and the bottompanel reveals the distribution of zinc in the identical voxels to thoseof the voxel matrix in panel B. In contrast to the two HMRs(⁵⁵Mn), onlya small region of HMR(⁶⁶Zn) emerges above the 2× median of the zincthreshold. Zinc, iron and copper values generally remain at those of thebulk tumour area in regions of HMR(⁵⁵Mn). This generalization does nothold in melanin-rich regions of melanoma tumours, where all four metalsare found together in high concentrations in concert with melaningranules.

It is acknowledged by the skilled addressee that there are a number ofmathematical and statistical methods of arriving at thresholds that areuseful in revealing regions of metallomic interest in tumours of mostuse for clinical decision making for radiotherapy.

FIG. 17 provides an example of the extent to which HMRs(⁵⁵Mn) occur indifferent tumour types, such as radiation sensitive versus radiationresistant ones. This comparison is of a 51 year old female patient witha small cell undifferentiated carcinoma of the lung, (Stage 1, T1N0M0)with the 54 year old patient who had a melanoma of the left forefinger(stage IV, T4N0M1). The region used herein for illustrative purposescorresponds to an approximately 1 mm by 1 mm sample of each of the twotumours, (31 by 31 voxels totaling 961 voxels). The eight panels of FIG.17 reveal areas of ⁵⁵Mn concentrations using four different thresholds,(i) T1, above a threshold of 0.5× the median value, (ii) T2, above athreshold of 1.0× the median value, (iii) T3, above a threshold of 1.5×the median value, and (iv) T4, above a threshold of 2.0× the medianvalue.

In the case of the small cell carcinoma of the lung, the voxels thatremain above the highest threshold of 2× the median value, T4, aregenerally non-contiguous ones (singletons) that are scattered throughoutthe sample. There are very few contiguous or adjacent concentrations ofHMR(⁵⁵Mn) voxels in this radiation sensitive tumour sample. In contrast,the four right hand side panels from the melanoma patient reveal theemergence of contiguous HMR(⁵⁵Mn) voxels that are readily discernible atthe T3, 1.5× the median value, and these HMRs(⁵⁵Mn) remain even at themore stringent T4 level of 2× the median value.

The data of FIG. 18 show, by way of non-limiting example, that when thesame threshold criteria are applied to the identical voxels of these twotumours, but now simultaneously assayed for HMRs(⁶⁶Zn), no HMR(⁶⁶Zn)areas are found at thresholds of T1, T2, T3 and T4 in the small cellcarcinoma of this particular patient and only a minor concentration ofcontiguous Zn voxels is apparent in the tumour of the melanoma patient.The distribution of ⁶⁶Zn voxel values remains relatively constant inboth these two tumours, while HMRs(⁵⁵Mn) predominate in the melanoma, asshown in FIG. 17.

This comparison of two tumour types, one radiation sensitive and theother radiation resistant is further highlighted by way of non-limitingexample, in the next seven Figures, (FIGS. 19 through 25). Theseillustrate the data from the 184 patients whose tumours are exemplary ofthe two spectral ends of radiation responsiveness: small cell lung,lymphoma and testis at the radio-sensitive end of the spectrum andmesothelioma, tumours of the brain and melanoma at the radio-resistantend of the spectrum.

By way of non-limiting example, an overview of tumour regions with highmetallomic concentrations and their clinical implications is providedbelow.

FIG. 19 shows the ⁵⁵Mn voxel data from each of twenty patients withsmall cell carcinoma of the lung when a tumour area of approximately1,800 voxels was sampled. The median ⁵⁵Mn value of the bulk of thetumour from each patient is represented by a square and is placed inbins of 200 consolidated counts per second over a range from zero CC/Sto 7,000 CC/S. All twenty patients have tumours where the bulk ⁵⁵Mnvalues fall below 3,000 CC/S, (grey squares). Fourteen of the twentypatients, shown by the grey squares above the top line, have noHMRs(⁵⁵Mn) when a T4 threshold of 2× median is used. By contrast, thetumours of six patients, t1 through t6, contain single HMRs(⁵⁵Mn) (blacksquares connected to the grey squares by a dotted line). In a specificexample, the bulk of the tumour of patient t6 has a median ⁵⁵Mn valuethat falls in the 2,400 to 2,600 bin, (grey square), but this tumouralso contains an HMR(⁵⁵Mn) whose value falls in the 6,400 to 6,600 bin(black square). The number of above threshold contiguous voxels of thesix HMRs(⁵⁵Mn) in patient samples t1 through t6 is 4.9%, 3.1%, 2.1%,12.3%, 12.6% and 5.2% respectively of the sampled tumour areas. In threeof these patients, t2, t5 and t6, the ⁵⁵Mn levels of their HMRs(⁵⁵Mn)exceed 4,000 CC/S. Such regions are likely to contain cells that exhibita degree of radio-resistance.

By way of non-limiting example a minimum size of 8×8 voxels efficientlyrevealed regions of high metallomic content. A person skilled in the artwill appreciate that below this size, and depending upon the sensitivityof the instrument used, smaller and smaller HMRs are picked up untilfinally they become indistinguishable from a randomly generatedbackground of singletons, as seen in the small cell lung carcinomas ofFIG. 17. An 8×8 HMR-containing landscape is bounded in both the X′ andY′ directions. It is contemplated that HMRs above X8×Y8 voxels canindividually increase by integers in the X′ and Y′ directions, yieldingHMRs ofX[8+1]×Y[8]:X[8]×Y[8+1]:X[8+1]×Y[8+1]:X[8+2]×Y[8]:X[8+2]×Y[8+1]:X[8+2]×Y[8+2]:X[8+1]×Y[8+2]:X[8]×Y [8+2] through X[8+n]×Y[8+n] where n is an integerthat can vary from 1 to thousands. Preferably, this integer varies from1 to 100, more preferably from 1 to 22, when a sample of 30×30 voxels (1mm² is sampled). A person skilled in the art will understand that thereis no impediment to setting a threshold lower than a minimum of 8voxels. The value of 8 has been used here because it isempirically-derived and serves as an example of an efficient search toolfor HMRs.

Sizes of HMRs

An important descriptor of any HMR is its size, shape and content.Tumours of the six types from a total of 184 patients were analyzed. Aperson skilled in the art will appreciate that HMRs can be variable insize, shape and content and that defining a minimal area will dependupon machine jitter, and in some instances a single voxel may representa rare, low frequency electronic “spike” in the data. In addition,machine “stutter” may occasionally produce two or three consecutive highvalues in the X or Y direction, but is readily recognizable byappropriate image filtering software.

In view of the above, an HMR for any metal [HMR(^(A)M), where ^(A)M=AnyMetal] may contain two adjacent voxels to fulfill the criterion of voxelcontiguity. Panel A of FIG. 20 shows the eight different theoreticalways in which two adjacent voxels (a doublet), can be configured from areference single voxel (starred). The size of HMR(^(A)M) can vary from2, 3, 4, 5, 6, 7, 8, 9, 10 etc contiguous voxels, to an integer which isthe sample size chosen for analysis of that particular tumour. A personskilled in the art will appreciate that size of HMR(^(A)M) according tothe invention is in any range measurable using existing technology. Forexample, HMR(^(A)M) size can range from 2 voxels to about 800,000 voxelsand any value in between (this being the maximum number of 35×35 micronpixels that can be analysed if a conventional slide is loaded to theedges with tumour material). A person skilled in the art will alsoappreciate that an HMR can apply to cancer cells in a tumour, or to aregion of cellular and non-cellular material, generally referred to asassociated stroma, which itself can exist in an “activated” state owingto its interaction with neighbouring cancer cells.

For a clinical application, a single sample size is typically an area of1 mm², as such a size has a precedent in routine pathological analysis.Thus when pathologists examine a tumour section for mitotic rate, forexample in melanoma, they have typically used the number of mitoses perhigh-power field, or per ten high-power fields (Burton et al., 2012, Am.J Surg. 204, 969-974). By way of non-limiting example, in respect ofHMRs(^(A)M), a 1 mm² sample conveniently approximates 1,000 voxels, inthis application. Multiple 1 mm² samples may be laser ablated from asingle tissue section on a glass slide. Multiple 1 mm² samples may belaser ablated from multiple sections from the same tumour block.Multiple 1 mm² samples may be laser ablated from multiple blocksrepresenting different samples from the same tumour, or its metastaticderivatives. The cumulative HMR(^(A)M) size determined from multiplesamples can vary from 4 voxels (2 doublets) to many millions for asingle tumour. It will be understood that the upper limit is constrainedby the practicality of the time required for analysis and health careexpense.

Shapes of HMRs

By way of non-limiting example, within a sample size of 1 mm², or ˜1000voxels of the 35×35×5 micron type, there can exist a large number ofHMR(^(A)M) shapes that conform to the condition of voxel contiguity. Asdescribed herein for the first time, a common shape is that shown byexample in the melanoma patient data of FIGS. 14 and 15 and representedstylistically in panels B and C of FIG. 20. Patterns such as thosedepicted in panels D, E, F, G and H manifest themselves in differenttumour types. For example, Panel D is one in which cancerous epithelialcells line a lymphatic vessel or duct, such as in breast and prostatecancer. Panels E and G would be representative of the classic “IndianFile” movement of cells which follow a structural motif in a tumour suchas a collagen bundle, or a major nerve tract in glioblastomas orastrocytomas, or the single file pattern seen in breast cancer sections.Panel F illustrates the case in which cancerous cells would be containedwithin a blood or lymphatic vessel, such as in Ductal Carcinoma In Situ(DCIS) in breast cancer. Panel H is representative of the distributionof cells and melanin deposits in some melanotic tumours.

It is understood that any known mathematical and statistical methods maybe used to analyze patterns and inhomogeneities in matrices and theassociated software. Such methods may reveal concentrations of voxels ofmany shapes and sizes, which may then be mapped onto the underlyingpathological landscape.

Footprints of HMRs(^(A)M) and their Value in Radio-Responsiveness

In a sample of 1,000 voxels using a 2× median threshold, the HMRs(^(A)M)(black squares in FIG. 20), panels B, C, D, E, F and G represent 4%,3.7%, 2.4%, 1.5%, 3.7%, and 0.9% of the sample.

As shown in FIG. 19, it is contemplated that if patients with tumourst2, t5 and t6 are treated with radiotherapy on either anintention-to-treat or palliative basis, the bulk of the cancer cells(with values <800, <1,800 and <2,600) would either be killed orsufficiently damaged as to be unable to undergo further cell division.By contrast, most of the cells of the HMRs(⁵⁵Mn) of these tumours (withvalues >4,000, >4,200 and >6,400) would survive and, in time, generate anew form of the tumour, giving rise to the familiar clinical finding oftumour reoccurrence.

HMR Data from Lymphomas, Tumours of the Testis, Mesotheliomas, the Brainand Melanomas

FIG. 21 shows the data from the 10 patients with diffuse B celllymphomas when a tumour area of approximately 1,800 voxels was sampledfor ⁵⁵Mn content by the Laser Ablation technology. None of the tumourshad HMRs(⁵⁵Mn) when a threshold of 2× median was used, as shown by theabsence of black squares. In addition, all median ⁵⁵Mn values fell below3,200 CC/S. The expectation is that if the tumours of these 10 patientswere treated with radiotherapy on an intention-to-treat or palliativebasis, the bulk of the cells would either be killed or sufficientlydamaged as to be unable to undergo further cell division. It is acceptedfrom everyday clinical practice in this area that large lymphoma massesare easily eradicated with radiotherapy.

FIG. 22 shows the data from the 55 patients with tumours of the testiswhen a tumour area of approximately 1,800 voxels was sampled. These 55tumours are all classic seminomas and form a unitary group on the basisof pathological criteria. Radiation oncologists and medical oncologistsconsider them to be very radiation sensitive, since large seminomamasses are easily eradicated with radiotherapy. Fifty one of theseseminomas have no HMRs(⁵⁵Mn) when a threshold of 2× median is used, asshown by the 51 grey squares above the top line. By contrast, fourpatients, S4, S5, S6 and S7, have single HMRs(⁵⁵Mn) (black squares)relative to their bulk values (grey squares). The sizes of these fourHMRs(⁵⁵Mn) ranged from 1.8 to 6.3% of the sampled area. In two of thesepatients, S6 and S7, the CC/S value of the HMRs(⁵⁵Mn) exceeds 5,000 CC/Sand such regions are likely to contain cells that are radiationresistant. These data support the probability when these 2 patients aretreated with radiotherapy on an intention-to-treat basis or palliativebasis, the bulk of the cells are killed or sufficiently damaged as to beunable to undergo further cell division, while the cells of theHMRs(⁵⁵Mn) survive and in time generate a new form of the tumour.

Finally, as was found from the earlier statistical analysis of theseseminomas, they are not a unitary group. It may well be that the tumoursof the three seminoma patients, 51, S2 and S3, which populate theextreme right hand tail of the top distribution, and whose bulk median⁵⁵Mn values are between 3,000 and 4,000, have a degree ofradio-resistance. The data from these three outliers (and from patientsS4, S5, S6 and S7, whose tumours do harbor HMRs(⁵⁵Mn)), are consistentwith the clinical data which reveal that a small proportion of seminomasexhibit signs of radiation resistance.

FIG. 23 shows the data from the 10 patients with mesothelioma when atumour area of approximately 1,800 voxels was sampled (with mesotheliomaconsidered to be a radiation resistant entity). Nine of the patientshave no HMRs(⁵⁵Mn) when a threshold of 2× median is used, as shown bythe grey squares above the top line. The one patient with the lowestmedian CC/S voxel value has a single HMR(⁵⁵Mn) whose CC/S exceeds 5,000(black square) and the size of which was 3.2% of the sampled area.Importantly, most mesothelioma ⁵⁵Mn values exceed those of the threesensitive tumour types.

FIG. 24 reveals the findings from the 25 patients with tumours of thebrain (glioblastomas and astrocytomas) when a tumour area ofapproximately 1,800 voxels was sampled (with tumours of the brainconsidered to be largely radiation resistant). Twenty-four of thepatients have no HMRs(⁵⁵Mn) when a threshold of 2× median is used, asshown by the grey squares above the top line. One patient with a lowmedian ⁵⁵Mn value has a single HMR(⁵⁵Mn) whose CC/S exceeds 6,000 andthe size of which was 3.6% of the sampled area. While most ⁵⁵Mn brainvalues exceed those of the three sensitive tumour types, these dataindicate that some glioblastomas and astrocytomas will respond toradiation treatment, as eight of these patients have median ⁵⁵Mn valuesbelow 3,000 CC/S.

FIG. 25 shows the data from the 64 melanoma patients, when a tumour areaof approximately 1,800 voxels was sampled, with melanoma historicallyconsidered to be a radiation resistant tumour. The melanomas revealextensive heterogeneity relative to the other five tumour types, with 29patients having tumours without HMRs(⁵⁵Mn), and 35 patients havingtumours with HMRs(⁵⁵Mn). Unlike the previous five tumour types whereHMRs(⁵⁵Mn) have existed as single entities within the tumour sample,half of the melanomas have multiple HMRs within the same tumour. Thesizes of the HMRs(⁵⁵Mn) vary from 1.6 to 21.8% of the sampled area.Owing to the stringency used herein for the threshold (2× median), thenumber of HMRs(⁵⁵Mn) per sample is actually a minimal estimate. FIG. 25also reveals that for nearly 30% of tumours, the median value of thebulk of a tumour falls below a CC/S threshold of 2K (grey squares) andfor two thirds of the tumours, the median value of the majority ofvoxels falls below a CC/S threshold of 4K (grey squares).

In hindsight, these atomic data indicate that many melanomas have areasof radio-sensitivity. In terms of metastatic disease, there aresubstantial differences in radio-responsiveness between cutaneous, lymphnode, visceral metastases or metastases to bone or brain. The widelyheld opinion of melanomas being radiation resistant is based onconflicting data, particularly evident by the findings that somemelanomas melt away after radiation treatment, while others can rapidlykill the patient in spite of radiation treatment. The metallomic datapresented herein, show for the first time, how traditionally conflictingdata can be partially resolved by the discovery of the previouslyunknown HMRs(⁵⁵Mn) in melanoma tumours.

FIG. 25 also shows that 88% of the melanoma HMRs(⁵⁵Mn) exceed a CC/Svalue of 4,000, (black squares) and nearly half of the HMRs(⁵⁵Mn) exceedvalues of 10,000 and some are near 100,000. These novel data indicatethat many melanomas may indeed initially be radiation sensitive, butsince most harbour HMRs which contain substantial levels of manganese,such cell populations would likely survive radiation treatment and thetumour would regenerate from radiation resistant remnants. The clinicaloutcome is that the patient returns post-radiotherapy with areoccurrence of the cancer, which is now more resistant than the initialtumour. Second, many of the melanomas contain large quantities of extra-and intra-cellular melanin, and as our data show, again for the firsttime in tissue sections analyzed by laser ablation, melanins cancontribute to radiation resistance by the biochemical processes outlinedbelow.

Melanins are heterogeneous polymers of uncertain 3D structure that formmultilayered complexes consisting of overlapping sheets ofdihydroxyindole and benxothiazine rings and sundry unidentified chemicalgroups (Zecca et al., Trends in Neurosciences, 26; 578-580, 2003). Inthe case of neuromelanin, there is also a large class of polyunsaturatedlipids. Neuromelanins act as sinks for many metals including chromium,cobalt, mercury, lead, and cadmium, and significantly for thisapplication, they also contain isotopes of Mn, Zn, Fe and Cu (FIG. 26).Melanins can be released extracellularly and, owing to their chemicalcharacteristics, can survive in the extracellular milieu for long timeperiods. Melanins have radio-protective properties, attested to bymelanized fungi living on the walls of the nuclear reactor at Chernobyland in the cooling water of currently operational nuclear reactors.Melanins scatter X-rays and act as a shield for radiation. Despite themassive literature on the properties of isolated melanins, the firstsystematic attempt to explain its radio-protective properties was onlymade in 2007 (Dadachova et al., Pigment Cell Melanoma Res. 21; 192-199,2007). Until the present invention, the physical explanation ofmelanin's radio-protective abilities at the level of pathological cancermaterial and their predictive relevance to patient treatment withradiotherapy has not been realized. Furthermore, most chemical andstructural studies to date have been based on isolated melanins, not ontheir in situ molecular properties, and there has not been a 2D analysisof tissue sections from tumors as described herein. This is highlightedby the examination of melanin concentrations in a melanoma from a 45year old female with a malignant melanoma of the chest wall (stage II,T4N0M0) (FIG. 27).

For clarity of presentation and for comparison of the concentrations ofdifferent entities, Panel A and panel B are duplicates of the same H&Estained section. The distribution of melanin within this section issufficiently high that staining by melanin specific antibodies has notbeen necessary to reveal it. The distribution of the four metals, ⁶⁶Zn,⁶³Cu, ⁵⁶Fe and ⁵⁵Mn, is shown in the four panels below, and can bevisually compared to the distribution of melanin.

Panel C shows the 2D distribution of ⁶⁶Zn, the highest concentration ofwhich (in black) co-localizes with the major melanin tracks. The bulkvalues for ⁶⁶Zn in the lightly stained areas are from 3,000 to 18,000CC/S, while the ⁶⁶Zn levels within the melanin tracks are from 18,000 to45,000.

Panel D shows the 2D distribution of ⁶³Cu (in black) which again closelytracks the melanin distribution. The bulk values for ⁶³Cu in the lightlystained areas are from 200 to 1,000 CC/S, while the ⁶³Cu levels withinthe melanin tracks are from 1,000 to 4,000.

Panel E shows the high concentration of ⁵⁶Fe (in black) which closelytracks the melanin distribution. The bulk values for ⁵⁶Fe in the lightlystained areas are from 20,000 to 80,000 CC/S, while the ⁵⁶Fe levelswithin the melanin tracks are from 80,000 to 100,000.

Panel F reveals the concentration of ⁵⁵Mn which closely follows themelanin tracks. The bulk values for ⁵⁵Mn in the lightly stained areasare from 1,000 to 2,500 CC/S, while the ⁵⁵Mn levels within the melanintracks are from 2,500 to 10,000. As the earlier example of FIG. 12showed, HMRs(⁵⁵Mn) that consist of heavily melanized regions, can reach⁵⁵Mn levels of 100,000. Melanin is thus a repository for high levels ofmetals that, if released, are likely to be a significant factor inradio-resistance. To our knowledge, this is the first demonstration ofin situ ⁵⁵Mn concentrations in melanin in a 2D tissue sectiondescription of cancerous tissue, and the first quantitative predictionsthat follow from such co-localization of metals and melanin for patienttreatment in radiotherapy. Melanomas have a dual defense to radiation.The first is the accumulation of ⁵⁵Mn in cells. The second is theproduction of large amounts of melanin, which not only act as a storagecapacitor for ⁵⁵Mn, but also likely provide some structural shieldingfrom ionizing radiation.

The sizes and shapes of the melanin-rich voxel tracks in FIG. 27 can becompared with the sizes and shapes of some possible voxel aggregationsas outlined in FIG. 20, (particularly in panel H of FIG. 20).

The melanomas are instructive in a further clinical sense, since thedata presented in FIG. 25 can be subdivided into melanoma samples thatare either from primary tumours, or ones that are from metastatic sites.These data are shown in FIG. 28. A comparison of the distribution ofmedian CC/S values for the different patients with primary or metastaticmelanomas yielded a D value of 0.1889 which has an associatednon-significant P value of 0.557. Thus in terms of the median ⁵⁵Mnvalues derived from a melanoma, the primary and metastatic melanomas areconsistent with being sampled from populations with the samedistribution.

A statistical comparison of the distribution of ⁵⁵Mn values from theprimary tumours and specifically from the metastatic ones that havealready spread to the lymph nodes, yielded a D value of 0.2222 which hasan associated non-significant P value of 0.442. Thus median ⁵⁵Mn valuesfrom primary tumours and those that have been sampled from lymph nodes,are consistent with being sampled from populations with the samedistribution.

An examination of the distribution of tumours with HMRs(⁵⁵Mn) and thosewithout HMRs(⁵⁵Mn) to determine if the frequency of HMRs(⁵⁵Mn) differsbetween primary and metastatic tumours reveals that they do not,(yielding a non-significant P value of 0.457). Thus in terms of thedistribution of median ⁵⁵Mn values, primary and metastatic tumours areindistinguishable. This finding is supported by there being no a prioribiochemical set of processes that would favour differential ⁵⁵Mnaccumulation between the primary tumour and its metastatic derivative(s)in lymph nodes.

While ⁵⁵Mn levels and their distribution within a 2D area in tumourtissue are exemplified, another metal, zinc, may also be helpful by wayof non-limiting example, as a discriminator in focussing on optimalregions for ⁵⁵Mn analysis. Many regions of a tumour contain cancerouscells intermixed with different cell lineages, as well as normalcellular and non-cellular components. The tumour milieu can containfibroblasts, extracellular matrix components, collagen bundles,capillaries, lymphatics and support cells such as pericytes and smoothmuscle cells, macrophages, osteoblasts, osteoclasts and components suchas hydroxyapatite in bone niches. If in the first instance it is cancercell populations that are chosen to be quantified for an ATI, then zinccan be used to differentiate between cells that are cancerous and cellsthat are not. Hence, zinc can provide a filter for ensuring that onlythe most relevant cancer cell-containing voxels are used for determining⁵⁵Mn values and their distributions. (Note that zinc does notdifferentiate between radio-sensitive and radio-resistant cells.) Zincvoxel values allow an initial avoidance of regions that may mislead inidentifying HMRs(⁵⁵Mn). This is illustrated in FIGS. 29 and 30.

Panel A of FIG. 29 illustrates an H&E stained section from a 54 year oldmale with a small cell undifferentiated carcinoma of the lung (StageIIIa; T2N2M0). Lightly stained regions of largely non-cancerous andnon-cellular material are apparent from the H&E view. Thislightly-staining region has low ⁶⁶Zn voxel values relative to thecancerous cells that are darkly staining. When an appropriate thresholdis applied to the ⁶⁶Zn data, the resulting image in panel B is anexcellent approximation to the differing regions seen in the H&E imageabove it. This means that the analysis of the ⁵⁵Mn values in this tumoursample is more appropriately based only on those ⁶⁶Zn voxels shown inpanel B, without a contribution from voxels of the non-cancerousregions. This is not meant to downplay the clinical significance ofstromal components, it serves simply to compartmentalize data analysis.This pre-screening of a tissue section landscape using ⁶⁶Zn, will beparticularly useful in tumours such as those of the breast and prostate,where non-cancerous material is much more intimately intermingled withcancer cell-containing voxels. The reciprocal of the above is that zinccan also be useful in determining which parts of a tumour can best yieldthe ATI of a stromal population.

A similar example is evident in FIG. 30, in which panel A illustrates anH&E stained section from a 66 year old male with a malignant melanoma ofthe rectum (Stage IIB; T4N0M0). Non-cancerous cell regions are againevident, particularly in the lower portion of the panel at 6 o'clock.When an appropriate threshold is applied to the ⁶⁶Zn data, the resultingimage in panel B mirrors the differing cellular components seen in theH&E image above it. This pre-screening with ⁶⁶Zn again shows thatsubsequent analysis of the ⁵⁵Mn values can be more accurately determinedusing only those ⁶⁶Zn voxels shown in panel B, without “contamination”from voxels of the non-cancerous/stromal regions.

It will be appreciated by those skilled in the art that other atomicelements and their associated isotopes, besides ⁶⁶Zn, may provide thesame useful function of differentiating between cancerous, normal,activated and non-cellular components of the stroma, particularly indifferent tissues.

Example 6 Cancers of the Breast

The data presented herein demonstrate for the first time that there is acorrelation between ⁵⁵Mn levels and the two spectral ends of tumoursthat relate to radiation sensitivity and radiation resistance. The datapresented herein also provide an insight into the previously unknownexistence of HMRs that are hidden from conventional pathologicalexamination and which play a major role in radiation resistance and anindication that a tumour may reoccur after radiation treatment. Inaddition, the data also provide a basis for metallomic contributionsthat derive from the interactions between stromal components and cancercell populations.

By way of non-limiting example, tumours that are loosely classified asbeing of “intermediate” radiation sensitivity, e.g., breast andprostate, were also analysed according to the invention.

The same quantitative approach of Laser Ablation—Inductively CoupledPlasma—Mass Spectrometry has been applied to 15 tumours of the breast,as was used for the six tumour types described herein. An overview ofthese data is presented in FIG. 31. Examination of the bulk ⁵⁵Mn valuesof these breast tumours (grey squares), indicates values between 1,000and 4,400 CC/S. At this level of inspection, they are indeedintermediate between the sensitive values found in the seminomas, smallcell lung and lymphomas, and the higher ⁵⁵Mn values of theglioblastomas, mesotheliomas and melanomas. However, over 70% of thesebreast cancers have HMRs(⁵⁵Mn) and their values extend from 4,000 to16,000 CC/S (black squares in the lower panel). In addition, sometumours have double and triple HMRs(⁵⁵Mn) using the stringent 8×8detection threshold. Until the present invention, it was not realizedthat the atomic data are consistent with the clinical observation that“breast cancers are very heterogeneous”.

In a personalized medicine context, the current medical art ofdescribing breast cancers as heterogeneous is not helpful in decidingwhich patients will benefit from radiotherapy and which patients shouldavoid the harmful effects of radiation treatment. The clinical realityis that in the absence of a useful predictive metric, and in thepresence of uniform H&E pathology, most breast cancer patients areirradiated after surgical resection of the tumour, e.g., in the USA, sothat all possible treatment modalities have been seen to be applied. Theresection margins for tumours of the breast can be large, so that thechance of surgically removing any residual cancer cells that may be atthe periphery of a tumour is increased. While radiation treatmentfurther increases the probability of destroying cells that have escapedresection, the harms of radiation could be avoided in those cases wherethe atomic data indicate very high levels of ⁵⁵Mn. In such high ⁵⁵Mncases, radiation treatment in an intent-to-treat situation is largelyfutile.

The top Panel of FIG. 32 illustrates a well differentiated invasiveductal carcinoma of the breast (Stage IIIa, T3N1M0) from a 39 year oldfemale. The H&E section is unremarkable and uniform by pathologicalinspection. By contrast, the atomic data seen in the bottom panel revealthe hidden heterogeneity of HMRs(⁵⁵Mn) (large black areas) and theirimportant clinical implications. This breast section has been examinedas an approximately 1 mm² area, to keep it in line with the standardsample size typically used in measuring mitotic rate.

FIG. 33 shows the analysis of an approximately 1 mm² area of 31×31voxels which was simultaneously analysed for the two atomic elements⁵⁵Mn and ⁶⁶Zn using the standard thresholds applied previously (T1, 0.5×median; T2, 1× median; T3, 1.5× median and T4, 2× median). TwoHMRs(⁵⁵Mn) become evident in the Mn analysis using the T4 threshold,while the identical voxels for ⁶⁶Zn remain at their bulk tumour levels.It is ⁵⁵Mn levels that are the critical characteristic of these HMRs. Apotential HMR(⁵⁵Mn) that has a size below the 8×8 threshold, is visibleabove the other two in panels T3 and T4.

Further analysis of this approximately 1 mm² area demonstrates theusefulness in analysing HMRs compared with bulk analyses of seeminglyhomogeneous pathological samples which “pool” data. Analysis of all 961voxels in this sample in histogram form is shown in FIG. 34 for ⁵⁵Mn and⁶⁶Zn values. In the top panel, the heterogeneity in high ⁵⁵Mn levels invoxels is seen as the peaks and the smear of values that are in excessof 5000 CC/S values and which reach a value around 15,000. Thisheterogeneity can be examined in a rigorous statistical manner e.g., byan analysis of voxel contents using Bowley's Non Parametric SkewStatistic (NPSS) (Bowley, 1901, Elements of Statistics, P S King andSon, publishers, Westminster, London, UK). By way of non-limitingexample, this was applied to the ⁵⁵Mn data and the NPSS value, [meanminus median]/[St.Dev] and found to be 0.25, whereas for the much moresymmetrical ⁶⁶Zn data it was found to be 0.02, representing a largedifference. It will be clear to a person skilled in the art that thereare a multitude of statistical approaches to measuring heterogeneity,which may be used with the Skewness shown herein.

The further area analysis revealed a distribution of the high ⁵⁵Mnvalues that consists of voxels spread uniformly throughout the sample,or their existence as aggregates. Finally, compared to the heterogeneityseen in ⁵⁵Mn levels, the lower panel shows that the identical Zn voxelswere more uniform in their values. This is correlated by the lack ofpeaks or a smear to the right of the major peak in the lower panel.

Cancerous Cells in Lymphatic Vessels/“Ducts” in the Process ofMetastasis

In contrast to the example of the morphological homogeneity of thepathology seen in the invasive carcinoma described above, the top panelof FIG. 35 illustrates the morphological heterogeneity of an invasiveductal carcinoma of the breast (Stage IIa, T2N0M0) from a 48 year oldfemale as visualized by H&E. It is an informative sample from theperspective of the ATI, as this section contains, (i) an area of normalbreast lobules at 3 o'clock (denoted N), with associated adipocytes,(ii) five areas denoted C1 through C5 in which cancer cells (denoted C),are visible inside lymphatic vessels and hence in the process ofmetastasizing, and (iii) an area of concentrated immune cells (denotedimmune), together with a stromal component, including adipocytes spreadthroughout the middle and right hand side of the sample.

The ⁵⁵Mn values of the normal lymphatic vessels, designated N, in thenormal portion of the breast, yielded CC/S values of 3,204, 3,104 and3,155. The lymphatic vessels that contain cancerous cells areinformative. Lymphatic vessel C1 yields a value of 2,904, C2 and C3yield 2,804 and C4 yields 2,771. Thus the ⁵⁵Mn contents of thesetransiting metastatic cells are little different from their progenitorcells constituting a normal duct of the breast. In contrast, thetransiting cells in lymphatic vessel 5 have a median CC/S of 8612 andare predicted to be radio-resistant. This example illustrates the atomicmicroheterogeneity that occurs in a small single sample of the breast.This microheterogeneity cannot be extracted from the pathology of thecancer cells in C1 through C5 which are in the process of metastasis,since at the microscopic level they appear indistinguishable.

The metallomic data reveal that all four metals, ⁵⁵Mn, ⁶⁶Zn, ⁶³Cu and⁵⁶Fe readily resolve the areas that consist of adipocytes as against thecellular and acellular regions seen in the H&E stained sections (FIG.36). The metallomic contents of adipocytes are diluted by the largeconcentration of lipids in such cells.

The heterogeneity issue, seen so clearly in this breast cancer sectionat both the morphological and metallomic levels, is also pervasive inother cancer types, even in different large regions within the sameorgan.

There are a number of important issues that are taken into account bymedical professionals regarding the treatment of a patient with cancer,including, but not limited to, the age of the patient; the currenthealth condition of the patient; comorbidities; the locations of thetumour(s) (primary or metastatic); whether surgery, chemotherapy, drugtreatment, immunotherapy or radiation is an option and whether thetreatment is made as intent-to-cure, or palliative. This listexemplifies the clinical decision network that must be navigated toyield the best options. The central decision-node after blood tests andscans have been completed, is the pathology of the tumours. All otherdecisions follow from the information obtained at this node, since aninference is made as to whether the tumour is likely to be benign orlikely to progress. Except in the case of measuring mitotic rate, untilthe present invention, the inference is currently not based onquantitative data, but on the general experience with a particulartumour type. For tumours that are relatively uncommon, there are usuallyfew clinical trials that provide guidance on which therapeutic step isthe best option. Even for common tumour types, such as those of thebreast, prostate, brain, skin and ovary, the shortcomings in themultitude of clinical trials that do exist have lead to continuingcontroversies. The major problem is that the data gathered at thispivotal pathological-decision node of a particular tumour type are notof requisite quantitative quality, and until this application, are notgeneralizable to all tumour types.

The clinical “value” of the crucial pathological information (namely apointer to whether a major therapeutic option such as radiation shouldbe implemented), rests largely on subjective interpretation of stainingmethodologies, combined with antibody information, or various forms ofin situ hybridizations, which are further complemented by newer genomicand proteomic technologies. As reinforced by further examples below, theatomic data provide a new measure of quantitation that has hitherto beenlacking at this key pathology/radiation-treatment, decision node. TheAtomic Therapeutic Indicator of any tumour type provides for the firsttime, a quantitative underpinning of which patients are suitablecandidates for radiotherapy, and in which patients a tumour is likely toreoccur after radiotherapy.

Example 7 Cancer of the Prostate

FIG. 37 shows a clinical example of the heterogeneity in pathologicalstatus in different parts of a tissue or organ, in this case theprostate gland. It is illustrated by a Final Prostate Biopsy Report fromthe USA (with the patients' full permission of disclosure). The patient(hereinafter denoted patient X), has adenocarcinoma of the prostratewith a PSA level of 8.1 ng/ml. The biopsy report documents theabnormalities in different regions of the prostate with 7 out of 12regions showing changes of little significance, and five regions with“cancer” as defined by Gleason scores no higher than 7 (upper panel).The diagnostic summary of the extent of involvement of each of thetwelve core biopsies is also shown (lower panel; Part A through Part L).Before the present invention, this patient would be assessed forradiation treatment without any quantitative measure that demonstratessuch treatment would be warranted. This patient underwent radiationtreatment on the basis of personal preference after discussions with hisprimary care physician, the other option offered to him being radicalprostatectomy. In accordance with the present invention, the chemicaldistribution of elements within each of these regions is measured, e.g.,the ⁵⁵Mn levels, 2D distributions and HMRs(^(A)M), is determined bylaser ablation. The results are analysed using available statisticalmethods and statistical theory, including calculating a measure ofcentral tendency, where the common measures of central tendency are themedian, arithmetic mean and mode. Any other measures of central tendencyknown in the art may also be used, including but not limited togeometric mean, medimean, winsorized k-times mean, K-times trimmed mean,and weighted mean, and the data may also be transformed prior tocalculating a central tendency. For example, the data are also testedusing non-parametric statistics, including the Kolmogorov-Smirnov test,which involves making no assumptions about the distribution of data.Other statistical methods include a combination of Bayesian and morestandard statistics, clearly set out by Lee and co-authors in DemystifyStatistical Significance-Time to Move on from the P value to BayesianAnalysis, (Lee, J Journal of the National Cancer Institute, 103, 2-32010) and Berry, Carlin, Lee & Muller. Bayesian Adaptive Methods forClinical Trials. Chapman and Hall/CRC Biostatistics series. ISBN9781439825488. 2010; Nuzzo, Nature, 506, 150-152. 2014).

Other statistical methodologies include those set out in: Talfryn etal., British Medical Journal, 316, 989-991, 1998; Sterne & Smith,British Medical Journal, 322, 226-231, 2001; Bland & Altman, BritishMedical Journal, 328, 1073, 2004. In a clinical setting, other methodsinclude: Rubinstein et al., Journal of the National Cancer Institute,99, 1422-1423, 2007; Krzywinski & Altman, Nature Methods, 10, 1041-1042,2013)

Upon completion of the statistical analysis, a decision is made on theradio-sensitivity or radio-resistance of the different regions of thetumour and the most appropriate treatment. For example, a tumour that isdeemed to be radio-sensitive at the bulk level and in its cancercell-laden HMRs(⁵⁵Mn) (if any), is irradiated. For one that isradio-resistant, or has large HMRs(⁵⁵Mn), radiation treatment is notrecommended. Selective radiation treatment is also recommended forspecific areas of a tumour that is radio-sensitive. For example, anyradiation treatment that selectively targets a specific area of a tumouris recommended. Such selective radiation treatments that are availableand known in the art are contemplated. A person skilled in the art isable to readily determine which selective treatment is warranted.

The same quantitative approach of LA-ICP-MS has been applied to 10tumours of the prostate as used for the previous seven tumour typesdescribed so far. An overview of these data is presented in FIG. 38.

Conclusion: No tumours had high HMRs(⁵⁵Mn) and their voxel values aregenerally at the low end of the ATI spectrum (i.e. low range CC/S) andhence, from this sample, the tumours are expected to be mostly radiationsensitive.

Example 8 Primary Melanoma of the Skin Metastatic to the Brain

FIGS. 39 and 40 reveal the detailed clinical data and their comparisonto quantitative atomic data from a 70 year old patient (hereinafterdenoted patient Y), who first presented with a primary melanoma and wassubsequently diagnosed with a brain tumour. The patient was treated withwhole brain radiation, sundry drugs, immunotherapy (prembrolizumab) andfinally underwent a stereotactic craniotomy. This example illustratesthe level of clinical detail that is required to match the atomic data,as well as the clinical baseline involved in placing an ATI into routinemedical practice. The patient has provided full permission ofdisclosure.

Patient Y was initially diagnosed with a primary melanoma of the upperback, with clear sentinel lymph nodes, as well as a number of basal cellcarcinomas and squamous cell carcinomas. A number of years later,following a fit, MRI scans revealed a cerebral neoplasm of the leftparietal lobe (FIG. 39, panel A, arrowed). Following radiationtreatment, MRI scans revealed the presence of residual material at thetumour site, with its associated ambiguities of interpretation (FIG. 39,panel B).

The detailed pathology report stated that the sections from the skin ofthe primary melanoma showed an ulcerated nodular melanoma with thetumour cells being positive for MelanA and negative for 34Be12, which isconsistent with melanoma (FIG. 39, panel C). The Breslow thickness was3.8 millimeters; Clark level 4; ulceration, 3 millimetres; percent ofdermal invasive tumour width, approximately 50%; dermal mitoses 9 permm² (which is considered to be a high mitotic rate; dominant cell types,naevoid and epithelioid; intravascular and lymphatic invasion was notseen and actinic/solar elastosis was mild.

The melanotic area was widely excised from the upper right back andexcisions of four sentinel nodes were carried out. Sections were treatedwith S100, HMB45 and MelanA to confirm the lesion as being a primarymelanoma.

While the primary ellipse revealed melanoma, no further tumour wasevident in the wider excision. The adjacent epidermis did revealreactive changes. Sentinel lymph nodes 1, 2, 3 and 4 revealed noevidence of further malignancy based on staining with H&E andimmunoperoxidases, while the status of node 3 was not reported.Following the excision, the patient was declared “clear of cancer” andafter 6 months showed no signs of recurrent melanoma.

Three years later the patient collapsed but recovered, yet exhibitedsigns of incoordination of the left leg when walking. Similar episodesof involuntary movement of the leg recurred up to 20 times per day andinitially they were accompanied by a strange sensation on the left sideof the head. A cerebral MRI revealed a 14 mm contrast enhancing lesionin the paramedian left parietal lobe around the precentral gyrus (FIG.39). The radiologist gave a differential diagnosis of a low gradeglioma, but it was possible that since the patient had a melanoma 3years previously, that the cranial lesion could instead be a recurrentmelanoma. This ambiguity is reflected by the limitations of MRI. Theradiologist concluded that “the nature of this lesion is uncertain. Itis likely to represent a neoplasm of the brain which is low gradeparticularly as there is no mass effect or oedema associated with thelesion. The findings on the diffusion weighted scan of the low signal onthe ADC (Apparent Diffusion Coefficient) map make it likely to be a lowgrade lesion such as a ganglioglioma, but also consider a DNET(Dysembryoplastic Neuroepithelial Tumor) or pleomorphicxanthoastrocytoma without any cystic component. A smalloligodendroglioma remains a possibility”.

It was recommended that neurosurgery not be undertaken at the time, withstereotactic radiosurgery being the appropriate option. Given that thepatient had been diagnosed with a primary melanoma, it was possible thatthe brain lesion was a metastatic melanoma to the brain. The patientunderwent radiotherapy for assumed metastatic melanoma with the totaldelivered radiation dose being 25 Gy and also began an immunotherapeuticregimen course of pembrolizumab 2 months after radiation therapy. Afurther month later an MRI revealed that the midline frontal metastasishad diminished in diameter from 16 mm to 11 mm. At face value, thetumour had sensitive and resistant components. The tumour became betterdefined with a thinner enhancing margin and with a more discretelyhypointense centre. Most of this reduction in tumour size is due toradiation and not the immunotherapeutic drug pembrolizumab, as tumourregression after one month of this drug treatment averages only 6%,(Hamid et al., 2013, New England Journal of Medicine, 369, 134-144).This patient's tumour reduced from diameters of 16 mm:16 mm:16 mm to 11mm:11 mm:11 mm. This is a reduction of (5+5+5)/(16+16+16), 31%, of which6% can be attributed to the drug, and 25% to radiation. This means that80% (25/31) of the tumour's initial regression was due to radiation. Afurther MRI scan 3 months later revealed a blush of peripheral contrastenhancement in the white matter adjacent to the tumour nidus, suggestingtumour progress since the last examination. The tumour was still at 11mm longest diameter 2 months later. An even later MRI revealed markedprogression around the tumour which now measured 23 mm in the longestdiameter (FIG. 39 panel B), however it was not possible to tell if thiswas radio-necrosis or progressive tumour. The patient also begantreatment with Avastin, and it was decided to proceed to craniotomy.Prior to craniotomy, a hyperintense centrally necrotic tumour was noted,approximately 18×33 mm transaxial and 33 mm superior oblique, featuresin keeping with a high grade tumour. The patient underwent astereotactic craniotomy to remove this left frontal lesion.

The histopathology of the resected brain tumour revealed 2 pieces of tanand brown ragged friable soft tissue measuring 15 mm×10 mm×5 mm and 9mm×6 mm×5 mm. There were very few and scattered devitalized “melanoma”cells with smudgy nuclei and no unequivocal evidence of residual viablemalignancy in this material. The material was negative for the melanomamarkers melanA and HMB45 and no definite pigment was seen. In theabsence of marker confirmation, there is insufficient evidence tounequivocally state that this was a metastatic melanoma or anindependent brain lesion, although the pathologist leaned towards amelanoma.

Atomic Analysis of the Primary Tumour Biopsy

As there were no biopsies of the brain lesion prior to radiation, theonly available evidence for tumour identity and radio-responsivenessstems from the primary tumour (FIG. 39, panels C, D and E). A standardunstained 5 micron tissue section was obtained (with all ethical, legaland patient consent issues fulfilled), and the entire area of FIG. 39panel C was laser ablated and then examined for the presence of nests ofcancerous cells amongst the normal stromal heterogeneity that is visiblein most of panel C.

The numerical data obtained from the laser ablation are shown in FIG. 40where five different regions of the primary tumour were analysed. Themedian ⁵⁵Mn value from all five tracks after background subtraction was3,747 CC/S (ATI) placing the primary tumour in an intermediate range ofradio-responsiveness. The clinical data are compatible with this valuesince the tumour was ablated with radiation.

As noted above, the patient was also treated with an immunotherapeuticregimen of pembrolizumab which targets PD-1, so it is a combination ofall these factors that has contributed to the final outcome. However, aspointed out above, 80% of the reduction in initial tumour size can beattributed to the radiation treatment.

Example 9

Tumour Status after Irradiation of a Visible Tumour

There is one source of tumours that are favourable for a morequantitative approach in terms of ATI and these are “externally visible”tumours whose status, progress, and condition after radiation treatmentcan be more readily measured than “internal” tumours. Patient Z is anexample of such a case (FIG. 41). For privacy reasons, this case servesonly as an illustration of the directly measurable and unambiguousoutcome of radiation of an externally visible tumour, and highlights thedifference between the ambiguities experienced with the internal tumourissues experienced with the brain lesion of patient Y.

Patient Z was radiation treated for a squamous cell carcinoma, (FIG. 41top panel), which after radiation treatment resolved almost completelywithin six months (bottom panel). Had a biopsy been available for atomicanalysis, a comparison of the clinical response and the ATI would havebeen useful.

Unlike other diagnostics which provide information on whether a patienthas a particular tumour type, but do not provide the next therapeuticstep tailored specifically for that patient, the ATI is applicable toall tumour types. ATI is pan-diagnostic. It is not restricted in themanner of a PSA test for example, where having obtained a value above 4ng/ml, the next question is; what is the therapeutic intervention? Is itradical prostatectomy, radiation (external beam, brachytherapy, orproton beam), watchful waiting, cryotherapy, or androgen deprivationtherapy? Unlike the ATI, the PSA test itself does not provide thetherapeutic pointer.

In the case of cancers of the breast, even if complete resection of atumour with wide margins is carried out, followed by chemotherapy,radiation treatment, hormone therapy and drug treatment with Herceptinand/or Avastin and/or immune checkpoint inhibitors and/or immuneagonists and/or vaccines, what is the probability that the tumour willreoccur if there is no information of the primary source as regards itspotential radio-responsiveness? If the breast tumour biopsy had asub-threshold ATI for example, then the probability of its reoccurrenceafter radiation would be lower than if the ATI of the biopsy was abovethreshold and if the tumour had one or multiple HMRs(⁵⁵Mn), from whichcells may have already migrated. If tumour cells have already spread tothe nearby lymph nodes from the breast, then measurements of the ATI(high or low) in these nodes will provide a clinician with an indicationof whether a more vigilant monitoring of the patient is required.

Similarly in the BRAF^(V600E) mutation in melanoma, and in many genomictests, the presence of a targetable “driver” mutation is inferred from acell sample or from circulating nucleic acids in the vasculature. Thisdoes not have the high value of a 2D visualization of the tumourlandscape, since the former are a pooled group of entities. This is acritical differentiator between the use of an ATI and a pooled sample,where in the latter, it is impossible to tell whether a high readingderives from the output of a small group of cancer cells or activatedstromal cells, or whether most cells in the sample contribute to thereading. The clinical implications for treatment are very different. Amelanoma patient treated with vemurafenib, for example, who has a smallnumber of cells in the tumour producing the altered protein, will hardlybenefit from treatment, whereas the drug will be far more efficacious ina melanoma patient where a large number of cells in the tumour areproducing a defective protein product. This distinction is difficult tomake unless a 2D landscape is available.

Example 10 Measurements Using Radiation Sensitizers/Synergizers ¹⁰Boron

The radio-responsiveness of a tumour is determined by measuring ⁵⁵Mn andits calibrated signals according to the invention, and theradio-responsiveness may also be influenced by the addition of asensitizer. In such a case, the success of Boron Neutron Capture Therapywill depend both upon the total ⁵⁵Mn calibrated signal and that of thesensitizer. Adding a sensitizer such as p-boronophenylalanine to atumour cell population that is high in manganese, may not be as usefulas adding it to a cell population that is low in manganese. In thisexample, the ATI for a tumour is determined using LA-ICP-MS, beforeusing Boron Neutron Capture Therapy.

A tumour sample is taken from a patient who has been previously infusedintravenously with an FDA approved sensitizer, e.g., a ¹⁰Boronderivative, such as p-boronophenylalanine, or the intravenous infusionof liposomes containing boron derivatives as previously described (Heberet al., Proc. Natl. Acad, Sci. USA, 111, 16077-16081. 2014), or boronnanoparticles as previously described (Petersen et al., AnticancerResearch, 28, 571-576, 2008). The tumour sample is then examined for the2D distribution of ¹⁰Boron to determine whether its levels anddistribution will be beneficial in terms of radiation. Simultaneously,or separately, the distribution and level of Mn is determined. Therelative amounts of ⁵⁵Mn and ¹⁰B determines the suitable tumours ofpatients for radiation.

Boron Neutron Capture Therapy (BNCT) is briefly described. A number ofexternal entities are known to make tumours more sensitive to radiatione.g., Boron, ¹⁰B. A thermal neutron is captured by the nucleus of ¹⁰Band the ensuing fission reaction yields ⁷Lithium as a recoil, an alphaparticle, a weak gamma-ray (0.5 MeV gamma photon), and 2.4 MeV ofkinetic energy. The ⁷Li ion and the alpha particle are classified ashigh linear energy transfer radiation and are highly destructive.

In studies in mice, subcutaneous injection of cells pre-incubated withboron nanoparticles into mice, which were then irradiated with neutronradiation, lead to longer survival, since the growth of tumours wasdelayed (presumably because the tumours with boron were made moresensitive to neutron irradiation) (Petersen et al., Anticancer Research,28, 571-576, 2008). A clinical trial for the treatment of head and necktumours has been initiated by the Boneca Corporation (ClinicalTrials.govidentifier; NCT00114790). In addition, a phase I/II clinical trial onArgentinian patients (with multiple subcutaneous metastases ofmelanoma), who have been treated with p-boronophenylalanine and neutronradiation yield an almost 70% response rate (Menendez P Appl. Rad. Isot.67, (7-8 Suppl.)S50-S53). 2009). Boron Neutron Capture Therapy has alsobeen used for non-small cell lung cancer (Farias et al., Phys. Med. 30,888-897. 2014).

2 Deoxy-D-Glucose, 2-DG

Other useful radiosensitizers include, for example, those as summarizedin Shenoy & Singh, Cancer Investigation 10, 533-551, 1992. These include2 deoxy-D-glucose, 2-DG, which is a close analog of glucose but withoutthe hydroxyl group in position 2.

2-DG is taken up avidly by those tumour cells that preferentially useglucose as available fuel, but upon phosphorylation by hexokinase, 2-DGis not further metabolized. Thus by competing with glucose uptake andsubsequent steps, 2-DG causes metabolic stress and renders cells moresensitive to radiation. In cell lines exposed to ionizing radiation andsimultaneously treated with 2-DG, radiation damage was increased insome, and the usual heterogeneity between cell lines was observed(Dawrkanath et al., Int. J Radiat. Oncol. Biol. Phys. 50, 1051-1061,2001). It is thought that the radiosensitization of some tumour cellpopulations occurs via disturbances in thiol metabolism (Lin et al.,Cancer Research, 63, 3413-3417, 2003).

In early phase I/II clinical trials on patients with advanced braintumours, the toxicity and feasibility of using 2-DG in combination withlarge fraction, 5 Gy, radiotherapy, (2-DG plus RT) was found to be welltolerated (Mohanti, B, Int. J. Radiat. Oncol, Biol. Phys, 35, 103-11,1996).

Treatment of glioblastoma multiforme patients with increasing oral dosesof 2-DG and radiation revealed that up to 250 milligrams/kilogram ofbody weight was well tolerated with no significant damage to the normalbrain. In addition, some of 60 patients in this cohort revealed mediansurvivals that exceeded those of patients that only receivedradiotherapy (Singh et al., Strahentherapie and Onkologie, 8, 507-514,2005). A summary of patient treatment and outcomes is found inDwarakanath, J. Cancer Research and Therapeutics, 5, 21-26, 2009).

Finally in nude mice with heterotropic pancreatic tumours, treatmentwith 2-DG plus radiation resulted in inhibition of tumour growth andincreased survival, compared to controls (Coleman et al., Free RadicalBiology and Medicine, 44, 322-331, 2008).

Testing a tumour for ⁵⁵Mn (and any other relevant metallomic data),prior to radiation, is therefore another practical application of thetechnology of the invention to radiosensitizers, not just 2-DG.

Immunotherapies

Radiation, and the antitumour immune responses that follow, form aninteracting system with an increased presentation of antigens on thesurfaces of cancer cells and the release of a host of proteins andpeptides (and metals bound to proteins and peptides), that influence theresponses of antigen-presenting cells. Thus primary tumours that havebeen irradiated (or distant metastatic growths within the sameindividual that have not been irradiated), can become sensitized toattack by various immune cells. The mechanistic bases for this increasedsensitivity are actively debated but remain unresolved, (Sharabi et al.,2015, Oncology [Williston Park] 2015, 29(5), pii:211304; Formenti, JNatl Cancer Inst. 105, 256-265, 2013). Melanomas that are highlyresistant to radiation have high levels of melanin (which store a hostof metals). In the context of the present invention, efficacy ofimmunotherapies is also addressed. Without being bound by any particulartheory, the extensive and very different metabolic properties ofradiation resistant cancer cells, versus sensitive ones, will not likelybe treated equally by the immune system, either before or afterradiation treatment. Thus radiation concomitant with immunotherapy;radiation preceding immunotherapy, or immunotherapy preceding radiation,will yield very different populations of cells within a tumour owing todifferential selection. In this example, a tumour is first characterizedby its ATI according to the method of any aspect, embodiment or exampleherein and a useful immunotherapy is then applied. Efficacy of theimmunotherapy and/or when to administer radiation treatment, e.g.,before, during or after immunotherapy is contemplated by performing themethod of the invention according to any aspect, embodiment or exampleherein. Different types of immunotherapy are thought to interactdifferently with the same type of radiation. For example, Sipuleucel-Tfor castration-resistant prostate cancer (a dendritic cell vaccinedesigned to induce immunity against prostatic acid phosphatase),ipilimumab (anti-CTLA4) for unresectable metastatic melanoma,pembrolizumab and nivolumab (anti-PD-1) for melanoma, nivolumab formelanoma and advanced squamous non-small-cell lung cancer, andtremelimumab and lirilumab, are likely to produce different responses toradiation than immunotherapies involving chimeric antigen receptor Tcells (CAR-T based immunotherapies). Checkpoint Blockade Immunotherapyin combination with stereotactic radiation delivery is underway for thetreatment of glioblastoma, but glioblastoma is being treated as aunitary entity. Accordingly, any glioblastoma is characterized accordingto the method of any aspect, embodiment or example herein and anevaluation is provided for patients that best respond toimmunotherapies.

Rose Bengal and Melanoma

Rose Bengal (4,5,6,7-tetrachloro-2′, 4′, 5′, 7′-tetraiodo-fluorescein)is an industrial chemical patented in 1882 that turns yarn and food red.When applied intra-lesionally to cutaneous melanomas, there can besignificant shrinkage of some tumour(s) (Thompson et al., MelanomaResearch, 18, 405-411, 2008; Thompson et al., Ann. Surg. Oncol. 22,2135-2142, 2015). While treated skin lesions decreased in volume afterRose Bengal intra-lesional treatment, some of the distant untreatedtumours in the same patient also shrank, indicating that an immuneresponse was likely involved. The addition of radiotherapy (RT) to RoseBengal (RB) treatment can further enhance tumour ablation, as shown for3 patients who underwent both therapeutic modalities, RB plus RT (Footeet al., Melanoma Research, 2010, 20, 48-51, 2010). Note however, thatradiation treatment of these patients was not based on any a prioriknowledge of the radio-sensitivity or radio-resistance of their multipletumours, as radioresponsiveness was not measurable prior to thisapplication. In fact, it was noted that “there is still no consensus onthe optimal dose and fractionation in melanoma” (Foote et al., MelanomaResearch, 20, 48-51, 2010).

Rose Bengal may be considered as an agent that has multiple modes ofaction: a sensitizer of cells to radiation, a sensitizer viaaugmentation of the immune system, an “additive cell kill” agent, or asynergizer. It is not possible to differentiate between these as themolecular mechanism(s) of these interactants is unknown, and the spatialarrangement of cancerous and stromal cells that have taken up RB remainsunknown. The clinician is thus left with the difficult task of themanagement of melanoma patients, particularly those with regionalmetastatic regions, such as local, satellite and in-transit recurrence.The current treatment guidelines predominantly include surgicalexcision, local ablation, intra-lesional chemotherapy and targeted drugssuch as vemurafenib. All of these are challenging “due to diseaseheterogeneity and frequent and persistent proliferation of lesions”(Thompson et al., 2015, Ann. Surg. Oncol. 22, 2135-2142, 2015).

The relevance of the current application to Rose Bengal and therapeutictreatment options, is that the Rose Bengal molecule contains 4 iodineatoms that are easily measured in tissue sections by LA-ICP-MS. Thus asection of any tumour that has been intra-lesionally injected by RoseBengal can be simultaneously analysed in each voxel for Iodine, ⁵⁵Mn orany other atom, prior to, and after radiation treatment, to determinewhich cell populations are susceptible to radiation. In this manner onecan more precisely target susceptible lesions. There are no data as yetto determine the relative clinical efficacy of RB followed by radiation,or radiation followed by RB. What is clear is that any tumour that isaccessible to intra-lesional injection of RB, can be analysed by themethods of this application to provide quantitative data onradiotherapeutic options.

By way of non-limiting examples, injection of RB into the prostate via amultiple core needle approach, and simultaneous Laser Ablation analysisof Iodine and ⁵⁵Mn from tissue sections, will provide information onwhich cell types of normal, cancerous and stromal populationspreferentially retain RB. These spatial distributions will enhancedecision making as regards radiotherapy.

Similarly, intra-lesional deposition of RB into breast tumour regionswill allow simultaneous Laser Ablation analysis of Iodine and ⁵⁵Mn fromtissue sections, and provide information on which cell types of normal,cancerous and stromal populations preferentially retain RB (as depictedfor ⁵⁵Mn, 66Zn, ⁵⁶Fe and ⁶³Cu in FIGS. 35 and 36 in this application).These spatial distributions will enhance decision making as regardsradiotherapy.

Example 11 Clinical Implementation of the Atomic Therapeutic Indicator

Analysis of the data from eight different tumour types has revealed anumber of findings that place the technological and clinical aspects ofATI into perspective. Not unexpectedly, the melanomas are different toall other epithelial tumours, since no other tumour types synthesizemelanin (unless it is a fortuitous activation of all pathways thatculminate in melanin production in an unrelated cell type, or the resultof cell fusion between immune cells and melanoma cells). In addition,melanomas derive from the initial embryological derivatives of neuralcrest cells, which are migratory cells that populate and set updifferent embryonic structures. Except for the initial migratory natureof germ cells, neural crest cells are the only other transitory celltype that migrates over long distances. As described herein for variousmelanotic tumours, melanins colocalize with the high concentrations of⁵⁵Mn, ⁶⁶Zn, ⁵⁶Fe and ⁶³Cu. Of these, ⁵⁵Mn is most likely to provideradiation protection by its ability to bind O₂.⁻, H₂O₂ and the highlydangerous hydroxyl radical OH. when ⁵⁵Mn is bound to various chemicalentities (FIG. 2). In addition, high melanin concentrations throughout atumour may provide a degree of physical shielding from radiation that isnot available to any other tumour type. The only melanomas that canreasonably be placed with all other tumour types, are those melanomasthat through the inactivation of steps leading to melanin production,are completely amelanotic. It is salient in this regard that themelanomas that have been examined herein, and which have unambiguouslylow concentrations of melanin pigment granules by visual microscopicalexamination (which is not quantitative), have low ⁵⁵Mn CC/S values(median voxel values of 1,939, 1,239, 817, 1,439, 1,278, 939, 1,617 and1,678 CC/S). These all fall below the 2K ATI threshold illustrated inFIG. 11. If this finding is subsequently confirmed quantitatively, thenthe amelanotic condition, in the absence of HMRs, may be indicative of alow ⁵⁵Mn ATI, and radiation sensitivity.

In a clinical context, one modus operandi for the application of ATI totumour biopsies are the flow diagrams shown in FIGS. 42 and 43. In theformer, easily accessed melanomas would be removed by surgery with wideexcision as exemplified by patient Y. Analysis of voxel contents viaBowley's Non Parametric Skew Statistic (NPSS) (Bowley, 1901, Elements ofStatistics, P S King and Son, publishers, Westminster, London, UK),would provide the first indication of homogeneity or lack thereof.Second, the conditions for homogeneity would be that, (i) the NPSSvalues for ⁵⁵Mn, ⁶⁶Zn, ⁵⁶Fe and ⁶³Cu would all be low, (ii) that thesample had no HMRs(⁵⁵Mn) in either the cancer cell or stromalcompartments and (iii) no overt melanisation. If the sample was found tobe homogeneous, its ATI would be above or below a chosen threshold ofclinical importance. As the primary tumour has been resected, such dataon homogeneity and above or below thresholds, would provide informationon the probable characteristics of any cells that had metastasizedearlier, such as the cranial metastasis in patient Y. Analysis ofresected lymph nodes from a patient who had undergone removal of theprimary melanoma, would be analysed in the same manner as above. An ATIwould provide guidance on whether to initiate radiation should ametastatic derivative of the primary tumour arise at a later time. If abiopsy from a metastatic site is available, then the flow diagram ofFIG. 42 starts anew.

The right hand panels of FIG. 42 illustrate the situation when a biopsyreturns a sample that is heterogeneous in terms of voxel characteristicsand when the tumour is resected. The heterogeneity will derive from anumber of contributing factors, (i) heterogeneity within the mainlandscape, (ii) the presence, size and content of HMRs(⁵⁵Mn) in both thecancer cell and stromal compartments, and (iii) the presence of melaningranules and the extent of intracellular melanin concentrations. Theevaluation of all HMRs(⁵⁵Mn), will indicate the probability ofreoccurrence of a distant tumour (if any cells had metastasized earlyprior to the resection of the primary tumour).

The flow diagram of FIG. 42 also applies to any biopsies that are takenfrom sites that are not amenable to excision by surgery, and where itneeds to be ascertained by determining an ATI, if radiation is a usefuloption as regards intent-to-treat by radiation.

For all other tumour types besides melanomas, the flow diagram of FIG.43 will lead to a clinical decision point on whether to use radiation,or to spare it. The biopsy will reveal either homogeneity orheterogeneity (the latter being an unavoidable grey area in a clinicalsense). The tumour will either be resectable or not, and radiation willbe used if the ATI derived from the absence of HMRs(⁵⁵Mn) is belowthreshold. If the tumour is heterogeneous and resectable, the ATI willbe an indicator of what to expect if distant metastases arise at a latertime in the patient's life. If the tumour is not resectable, then thedecision to irradiate or not, is made on the basis of threshold values.

It will be understood by the person skilled in the art that a number ofother factors unrelated to the ATI will be considered by both theattending physician and the patient. These will include, but are notlimited to, the age of the patient, the current health condition of thepatient, comorbidities, the locations of the tumour(s) (primary ormetastatic) and any hereditary conditions that make a patientradiation-sensitive. In the case of brain lesions, some tumours will bemore radiation resistant than other tumour types, because the cancercells use a novel mechanism of interconnection via microtubes, whichallows damaged cancer cells to be repaired by others within the tumour(Osswald et al., Nature, 528, 93-98, 2015).

Example 12

Clinical Implementation of the Atomic Therapeutic Indicator inCombination with Other Entities

The key foundation of this application is that focused pulses of highirradiance laser energy applied across a tissue section, and theanalysis of the vaporized material via mass spectrometry, provide a 2Dspatial atomic map which is of immediate therapeutic importance asregards radiation treatment of cancer patients via an ATI. This ATI/H&Emap is the foundation onto which other different maps can besuperimposed. A person skilled in the art will recognize that ajudicious choice of biological entities providingmultilayered/superimposed information, will further increase theclinical impact of an ATI, a situation that was not available prior tothis application. We demonstrate below how integrating additional mapsemploying metal-labelled antibodies using elemental analysis, namelylaser ablation-Inductively coupled plasma-mass spectrometry (LA-ICP-MS)or laser ablation-time-of-flight-mass spectrometry (LA-TOF-MS) orinductively coupled plasma-optical emission spectroscopy (ICP-OES), ormicrowave plasma-atomic emission spectroscopy (MP-AES), or laser inducedbreak down spectroscopy (LIBS), or secondary ion mass spectrometry(SIMS), or X-ray absorption near edge structure (XANES), atomicabsorption spectroscopy (AA) or X-ray fluorescence (XRF), can increasethe power of the clinical decision making process.

Additional Maps

Gene expression can be measured in tissue sections via “spatialtranscriptomics” using arrayed reverse-transcription oligo (dT) primersand fluorescently labelled nucleotides (Stahl et al., Science, 353,78-82, 2016). This provides a spatial map of gene expression relative tothe H&E map of the tissue section, but it comes at the cost of beinglabour intensive, involving library construction, amplification steps,intensity loss of fluorescence with time, staining artefacts andauto-fluorescence as well as deconvolution of large data sets ofEntities of Unknown Clinical Significance. To our knowledge, no previousspatial gene expression maps of tissue sections have reported on theclinical question of whether radiation is a preferred treatment optionfor a patient.

What has not been available, until the present application, is aclinically useful 2D map generated from a simultaneous measurement of anATI together with specially selected biological parameters at theprotein or cellular level. These parameters need to have a presumedinvolvement in radiotherapeutics and need to be immediatelyimplementable with current pathological and molecular technologies. Anumber of instantiations of such maps are provided below.

Many tumours are claimed to contain “cancer stem cells” (CSCs) (Clevers,Nature Medicine, 17, 313-319, 2011), that are virtually resistant toradiation (Ogawa et al., 2013, Anticancer Research, 33, 747-754). Thisradio-resistance of CSCs is thought to be due to a number of factorsincluding their superior DNA repair capabilities and their heighteneddefence to Reactive Oxygen Species. Such CSCs are thought to self-renew,divide slowly and are capable of reconstituting a tumour. If such isindeed the case, then it would be clinically advantageous to constructand to superimpose a “properties of cancer stem cells” map, onto theATI/H&E map. This can be done using metal-labelled antibodies.

Current technology on formalin-fixed paraffin-embedded tissue sectionsgenerally employs antibodies to the antigens of interest, but tomultiplex several protein tumour markers, say 4 or 5, which mayco-localize on the same tissue section, is near impossible using currentimmunohistochemistry. Use of a primary antibody which is antigenspecific, is followed by an amplification step which involves an enzymelabelled secondary antibody. The time factor of staining 5 sequentialtissue sections, processed at different times and different stainingconditions, is neither conducive to rapid and accurate throughput, norto interpretation. However, application of antibody labelling usingmetals (especially lanthanides and their easily distinguished isotopes),means that different antibodies, each tagged with a different isotope,can be applied to the same tissue section which is then directlyexamined via LA-ICP-MS, (Giesen et al., 2011, Anal. Chem. 83,8177-8183). Here there is no ambiguity with co-localized stainingprocedures, fluorescence issues or quantification. This methodology hasbeen applied in a diagnostic context to the labelling of primaryantibodies, anti-Her2, anti-CK-7 and anti-MUC 1 using the lanthanidesholmium, thulium and terbium and their subsequent location in breastcancer tissue sections examined via LA-ICP-MS, (Giesen et al., 2011,Anal. Chem. 83, 8177-8183). It has also been applied to directly labelanti-tyrosine hydroxylase (TH) with Ytterbium-173, Paul et al., 2015,Chemical Science, DOI:10.1039/c5sc02231b, 2015).

The above data demonstrate that multiple lanthanide-labelled antibodiesanalysed via elemental analysis can report on the spatial distributionof antigens in the same tissue section and provide clinical informationof use for drug-based patient treatment. In the context ofradiotherapeutic information, however, the requirement is different. Itis to measure an ATI and entities of radiotherapeutic significance inthe same tissue section, or in sequential serial sections (for examplein the breast cancer section exemplified in FIG. 35, where there arecancerous cells in lymphatic vessels, and where those metastasizingcells have different ⁵⁵Mn levels). This can be implemented as follows.

It is known that the 15 lanthanides; Lanthanum, Cerium, Praseodymium,Neodymium, Promethium, Samarium, Europium, Gadolinium, Terbium,Dysprosium, Holmium, Erbium, Thulium, Ytterbium and Lutetium, togetherwith Scandium and Yttrium, have multiple isotopes that are readilydistinguished by LA-ICP-MS. Hence instead of 17 elements, one has aminimal palette of around 32 labels from which to choose to labelantibodies of choice. In the context of this application, the next stepis to select those antigens that are markers for “stemness”, DNA repair,ROS, cell division and methylases and demethylases involved in silencingof tumour suppressors in cancer cells, and all of which impinge onradiotherapeutic relevance.

In a non-limiting context, the first steps involve:

(i) selecting proteins involved in “stemness” networks that are relevantto tumour aggressiveness/metastatic potential, such as CD44, (breast,liver and pancreas), CD133 (brain, colorectal, lung, liver), EpCAM,(colorectal and pancreatic) as set out by Clevers (Nature Medicine, 17,313-319, 2011), as well as proteins that are targeted by drugs aimed atcancer stem cells such as NOTCH, DLL4, FAK, STAT3, and NANOG (Kaiser,Science, 347, 226-229, 2015).(ii) selecting proteins involved in DNA repair, such as BRCA1, BRCA2 andATM, as reviewed by Wood et al. (2001, Science 291, 1284-1289, andsubsequent updates).(iii) selecting proteins involved in metabolic networks of ReactiveOxygen Species which have a major role in the tumour niche, such asHypoxia Inducible Factors, HIF (Simon and Keith, Nature ReviewsMolecular Cell Biology, 9, 285-296, 2008), carbonic anhydrase IX andcatalase.(iv) selecting proteins involved in cell division networks, such asKi-67 (Inwald et al., Breast Cancer Res Treat. 139, 539-552, 2013).(v) selecting proteins such as the DNA demethylase TET1 and TET 3(Forloni et al., Cell Reports, 16, 1-15, 2016), and DNA methylases suchas the DNMT1, DNMT3a and DNMT3b that silence or activate genomic regionsand influence the oncogenic potential of cells within tumours.

The second step involves using primary antibodies to these proteinproducts and then labelling either primary or secondary antibodies witha suitable lanthanide and hybridizing these antibodies to tissuesections.

The third step involves the application of elemental analysis to such anantibody-rich section. This provides a simultaneous readout of theendogenous metals that have been used in this Application, Mn, Zn, Feand Cu, to generate an ATI and to locate HMRs, plus a spatial readout ofthe various lanthanides that highlight the positions in the ATI/H&E mapof the relevant proteins to which the lanthanides are attached. This ismultiplexed rapid cartography of clinical importance. When applied to atissue section such as that of FIG. 35, it will allow for a morecomplete understanding of the characteristics of the cell populations inlymphatic vessels and capillaries of the breast (and any other tumour),the heterogeneity within primary and secondary tumours, and in the caseof core needle biopsies of the prostate, an expansive view that farexceeds that of current pathological insights.

This logical extension of superimposed multilayered information onto anATI/H&E map, provides a new pathological taxonomy, not previously seen,that produces rapid, quantitative, clinically relevant information fromthe same or serial tissue sections which can be evaluated in aradiotherapeutic context.

Advantages of the Present Invention

The 2D cartographic nature of the present invention has a number ofsignificant attributes that are relevant to the clinicalradiotherapeutic treatment that can be undertaken compared to thenon-cartographic alternative of homogenizing a tumour and measuring theamount of manganese per unit mass of tissue.

-   -   First, all tumours are heterogeneous in terms of the amount and        type of stromal material, so the relative amount of tumour cells        to stromal material, which is only visible and measurable with        reference to a 2D map, will influence the median ATI.    -   Second, even when a 2D region consists of nearly all cancer        cells, they can be very different in terms of their Mn levels.        FIG. 1 illustrates this perfectly, where a third or so of the        section consists of amelanotic tumour cells with median CC/S        values around 3,000, and the rest of the section consists of        areas where the Mn levels vary between 15,000-45,000 CC/S and        then HMRs(⁵⁵Mn) that vary between 45,000-150,000 CC/S. If one        had homogenized this section, the median ATI would far exceed        10,000 CC/S. What would have been lost in a clinical sense, is        that radiation treatment of this patient would have killed the        third of the tumour that has a low ATI, and thus the tumour        burden would have been reduced, with clinical benefit to the        patient.    -   Even more importantly, homogenizing a section, or a tumour        sample, loses one of the important aspects of the invention:        HMRs(⁵⁵Mn) that are predictive of tumour reoccurrence. Most HMRs        represent less than 10% of a section, so homogenization loses        that information of the presence of an HMR(⁵⁵Mn), as the median        ATI is barely moved.

In the example of breast cancer, FIG. 35, with its mixture of normalbreast ducts, an area of immune cells, adipocytes and stromal cells, theclinically relevant cells that are most dangerous are the ones that arealready in only one lymphatic vessel C5. They are in the process ofmetastasis and they have a median of 8,612 CC/S. If the entire sectionwere homogenized, this clinically relevant information is completelylost, as the median of the homogenized section is now approximately1,669 CC/S, a low value that is influenced by the proportion of theadipocyte population, the stroma and the relatively low values in thenormal ducts.

1-31. (canceled) 32-56. (canceled)
 57. A method of determining theradio-responsiveness of a cancer, the method comprising generating anAtomic Therapeutic Indicator (ATI) of a test sample from the cancer;wherein the method of generating the ATI comprises quantifying the levelof manganese in voxels in a 3D region of the test sample, the methodcomprising: (a) selecting a 2D region of said test sample, wherein the2D region is topographically defined by an X′:Y′ coordinate systemwherein X′ is the length of the 2D region and Y′ is the breadth of the2D region, wherein the 3D region corresponds to said 2D region and has aselected height represented by Z, wherein the 3D region is divided intovoxels of a pre-defined volume, the volume of each voxel being definedby X×Y×Z wherein X is the length of the voxel, Y is the breadth of thevoxel, and Z is the height of the voxel; (b) quantifying the level ofmanganese in each voxel; and (c) calculating the central tendency levelof manganese in selected voxels; wherein the central tendency level ofmanganese in the selected voxels defines the ATI; wherein the lower theATI the more sensitive the cancer is to radiation and the higher the ATIthe more resistant the cancer is to radiation.
 58. The method accordingto claim 57, wherein quantification of the level of manganese in thevoxels of the test sample is calibrated using a reference standard,wherein the reference standard comprises one or more reference voxels,and wherein each reference voxel comprises a known quantity ofmanganese.
 59. The method according to claim 58, wherein referencestandard is a biological sample comprising a known quantity ofendogenous or exogenous manganese.
 60. The method according to any oneof claim 57, wherein the ATI is expressed in calibrated counts persecond (CC/S).
 61. The method according to any one of claim 57, whereinthe central tendency level is the median, arithmetic mean, or mode. 62.The method according to claim 57, wherein the level of manganesequantified in a 3D region of one or more control sample(s) is quantifiedconcurrently, or sequentially in any order, when the test sample isquantified, or side-by-side with the test sample.
 63. The methodaccording to claim 57, wherein the ATI is compared to a pre-determinedATI threshold wherein the radio-responsiveness of the cancer isdetermined by assessing whether the ATI is above or below the ATIthreshold, and wherein if the ATI is below the ATI threshold the canceris determined to be sensitive to radiation; and wherein if the ATI isabove the ATI threshold the cancer is determined to be resistant toradiation.
 64. The method according to claim 57, wherein the ATI iscompared to two pre-determined ATI thresholds wherein theradio-responsiveness of the cancer is determined by assessing whetherthe ATI is above or below the two thresholds, and wherein if the ATI isbelow the lower ATI threshold the cancer is determined to be sensitiveto radiation; wherein if the ATI is above the second ATI threshold thecancer is determined to be resistant to radiation; and wherein if theATI is between the two ATI thresholds the cancer is determined to bepartially sensitive to radiation.
 65. The method according to claim 57,wherein the selected voxels are voxels in which cancer cells aredetected.
 66. The method according to claim 65, wherein the cancer cellsare detected by visual inspection of the 2D region of the test samplestained with a stain that distinguishes cancer cells from other cells,and preferably the stain is hematoxylin and eosin (H&E) stain.
 67. Themethod according to claim 65, wherein the cancer cells are detected bybinding of an antibody, preferably a metal-labelled antibody, to thecancer cells.
 68. The method according to claim 57, wherein X is in anyrange measurable and preferably in the range of about 1 micron to about200 microns.
 69. The method according to claim 68, wherein X is selectedfrom about 10 to about 50 microns and any value in between, andpreferably X is about 35 microns.
 70. The method according to claim 57,wherein Y is in any range measurable and preferably in the range ofabout 1 micron to about 200 microns.
 71. The method according to claim70, wherein Y is selected from about 10 to about 50 microns and anyvalue in between, and preferably Y is about 35 microns.
 72. The methodaccording to claim 57, wherein Z is in any range measurable andpreferably in the range of about 1 micron to about 20 microns.
 73. Themethod according to claim 72, wherein Z is selected from about 1 toabout 20 microns and any value in between, preferably Z is about 1, orabout 2, or about 3, or about 4, or about 5 microns.
 74. The methodaccording to claim 57, wherein X, Y and Z are selected respectively inranges from about 1 to 200 microns, 1 to 200 microns, and 1 to 20microns and any value in between in each range.
 75. The method accordingto claim 74, wherein X, Y and Z are 35 microns, 35 microns and 5 micronsrespectively.
 76. The method of claim 57, wherein the pre-defined volumeof each voxel is in the range of about 1 cubic micron to about 8×10⁵cubic microns, or about 1 cubic micron to about 10,000 cubic microns, orabout 2000 cubic microns to about 8,000 cubic microns.
 77. The method ofclaim 76, wherein the pre-defined volume is about 6,125 cubic microns.78. The method according to claim 57, wherein quantification of thelevel of manganese is in the voxels is by laser ablation-Inductivelycoupled plasma-mass spectrometry (LA-ICP-MS), laserablation-time-of-flight-mass spectrometry (LA-TOP-MS), inductivelycoupled plasma-optical emission spectroscopy (ICP-OES), microwaveplasma-atomic emission spectroscopy (MP-AES), laser induced break downspectroscopy (LIBS), secondary ion mass spectrometry (SIMS), X-rayabsorption near edge structure (XANES), atomic absorption spectroscopy(AA) or X-ray fluorescence (XRF).
 79. The method according to claim 57,wherein the voxels are arranged in a track across the test sample, andquantifying the level of manganese is carried out by laser ablation ofthe track.
 80. The method according to claim 57, wherein the test sampleis selected from a cell, a population of cells, one or more singlecelled organism(s), a tissue sample, or part thereof, an organ sample orpart thereof, one or more cells obtained/derived from a prokaryotic oreukaryotic organism, a population of cells and its associatednon-cellular stromal components, a neoplastic cell or population ofneoplastic cells, a tissue sample from any organ or tissue from asubject, a tumour, a solid mass or a “liquid” population of cells, cellsof any cancer of the hematopoietic system including leukemic cells, thecirculating cellular derivatives of solid tumours, and a cell orpopulation of cells that has metastasized.
 81. The method of treating acancer in a subject comprising performing the method of claim 57 on atest sample from the subject, and including radiotherapy in treatingsaid cancer in the subject if the test sample is determined to besensitive to radiation.
 82. The method of treating a cancer in a subjectcomprising, performing the method of claim 57 on a test sample of thesubject, and not administering radiotherapy in treating said cancer inthe subject if the test sample is determined to be resistant toradiation.
 83. The method according to claim 82, wherein the subject isa human.
 84. The method according to claim 62, wherein the controlsample comprises or is derived from a cell, a population of cells, oneor more single celled organism(s), a tissue sample, or part thereof, anorgan sample or part thereof, one or more cells obtained/derived from aprokaryotic or eukaryotic organism, a population of cells and itsassociated non-cellular stromal components, a neoplastic cell orpopulation of neoplastic cells, a tissue sample from any organ or tissuefrom a subject, a tumour wherein the tumour for example, is a solid massor a “liquid” population of cells, any cancer of the hematopoieticsystem including leukemic cells, the circulating cellular derivatives ofsolid tumours, or a cell or population of cells that has metastasized.85. The method according to claim 57, wherein the test and/or thecontrol sample comprises or is derived from a cell, a population ofcells or a tissue sample of a tumour/neoplasm of breast cancer, prostatecancer, cancer of the testes, lymphoma, small cell lung cancer, cancerof the brain, mesothelioma or melanoma.
 86. A method of determining thelikelihood of reoccurrence of a cancer in a subject after a radiationtreatment to the subject, the method comprising: (a) quantifying thelevel of manganese in a 3D region of a test sample from the cancer byselecting a 2D region of said test sample, wherein the 2D region istopographically defined by an X′:Y′ coordinate system wherein X′ is thelength of the 21) region and Y′ is the breadth of the 2D region, whereinthe 3D region corresponds to said 2D region and has a selected heightrepresented by Z, wherein the 3D region is divided into three or morevoxels of a pre-defined volume, the volume of each voxel being definedby X×Y×Z, wherein X is the length of the voxel, Y is the breadth of thevoxel and Z is the height of the voxel, and quantifying the level ofmanganese in each voxel; (b) identifying in the 2D region correspondingto the X and Y coordinates of the voxel, high metallomic regions (HMRs),being regions of the cancer in which the level of manganese is higherthan in the surrounding areas as enabled by statistical thresholds thatare multiples of a central tendency or any approximation betweenintegers; wherein the higher the frequency of HMRs the higher thelikelihood of the cancer reoccurring and the lower the frequency of HMRsthe lower the likelihood of the cancer reoccurring.
 87. The methodaccording to claim 86, wherein the HMRs are also identified in the 2Dregion of the test sample stained with a stain that distinguishes cancercells from stromal components, and preferably wherein the stain ishematoxylin and eosin (H&E) stain.